Recombinant modified saRNA (VRP) for cancer vaccines

Abstract

The present invention provides self-amplifying RNA (saRNA) for use in treating tumors. Treatment is provided by using saRNA, particularly VRP, containing nucleic acids encoding tumor-associated antigens (TAA) and IL-12. In some embodiments of the present invention, methods include intratumoral injection of these saRNAs. In some embodiments, the saRNA is injected intraperitoneally to stimulate an immune response against peritoneal tumors.

Description

[Technical Field]

[0001] Provided herein are cancer vaccines, which in one embodiment are administered to a subject in need thereof. The vaccine comprises a nucleic acid molecule that provokes a protective response against cancer in a patient. In one embodiment, a vaccine is provided, which can be delivered by self-amplifying RNA (saRNA), for example, by an alphavirus replicon (VRP), and in a preferred embodiment, an alphavirus vector replicon particle vaccine is administered to a patient. Another embodiment provides a saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a nucleic acid encoding IL-12. Accordingly, the present invention also relates to compositions comprising a saRNA, for example, a VRP encoding IL-12, and a tumor-associated antigen (TAA). The present invention also relates to vaccination methods, particularly, allogeneic prime-boost vaccination regimens using two viral vector compositions. More specifically, the present invention relates to recombinant VRP (saRNA) for use in allogeneic prime-boost vaccination regimens. The present invention also relates to products, methods, and uses thereof, suitable for, for example, inducing a protective immune response in a subject. [Background technology]

[0002] Vaccines are one of the most effective, safe, and economical strategies for disease prevention and disease spread control. Traditional vaccines are a form of immunoprophylaxis administered before disease onset, providing immune protection by generating strong host immune memory against specific antigens. The primary goal of vaccination is to activate adaptive-specific immune responses, primarily by generating B and T lymphocytes against specific antigen(s) associated with the disease or pathogen.

[0003] Similarly, cancer vaccines aim to induce an immune response against cancer tumor-associated antigens. Cancers are immunogenic and can activate host immune responses capable of controlling disease and causing tumor regression. However, cancers also have specific and nonspecific immunosuppressive effects, allowing them to evade the host immune system. Many protein / glycoprotein tumor-associated antigens have been identified and associated with specific types of cancer. Her-2-neu, PSA, PSMA, MAGE-3, MAGE-1, gp100, TRP-2, tyrosinase, Mart-1, β-HCG, CEA, Ras, B-catenin, gp43, GAGE-1, BAGE-1, MUC-1, 2, 3, and HSP-70 are just a few examples.

[0004] Naturally derived and recombinant cancer protein antigen vaccines are subunit vaccines. Unlike whole-cell vaccines, these subunit vaccines contain defined immunogenic antigens at standardized concentrations. The greatest challenge in developing such vaccines is finding the appropriate adjuvant and delivery system. Furthermore, purifying natural or recombinant tumor antigens is laborious and not always logistically feasible. Protein cancer vaccines require tumor cell culture, tumor antigen purification, or the production of specific peptides or recombinant proteins. Furthermore, vaccines made solely from tumor proteins / peptides have inherent problems. They may be limited in their ability to direct the appropriate antigen presentation pathway or may not be recognized by the host due to host major histocompatibility complex (MHC) polymorphism. Vaccines containing nucleic acids encoding tumor antigens, unlike vaccines containing the antigen itself, address some of these issues. To date, these approaches have shown the most promising results in preclinical and clinical trials. Of the technologies currently being applied to cancer vaccines, two systems in particular show great potential for application in this field. The first is the delivery of TAAs using viral vectors, including but not limited to adenoviruses, adeno-associated viruses, retroviruses, poxviruses, flaviviruses, picornaviruses, herpesviruses, and alphaviruses (see WO 99 / 51263). The second is vaccination with tumor cell proteins or RNA using exogenously derived dendritic cells as a delivery vehicle for the transfer and expression of TAAs into the host (Heiser et al., 2002. J. Clin. Inv. 109:409-417 and Kumamoto et al., 2002. Nature Biotech. 20:64-69).

[0005] To date, naked DNA, RNA, viral, and bacterial vectors have been evaluated for their ability to induce cancer-specific responses against tumor antigens. Attempts to enhance the immune response elicited by naked nucleic acid vectors include the use of self-replicating viral vectors delivered in the form of naked RNA or DNA (Ying et al., 1999, Nature Medicine, 5:823-827).

[0006] Alphavirus vector delivery systems have been identified as attractive vaccine vectors for several reasons, including high expression of heterologous gene sequences, induction of highly safe non-replicating (alpha)virus replicon particles (ARPs), an RNA genome that replicates in the cytoplasm of target cells, eliminating the possibility of vector integration into the genome, and finally, the demonstrated ability of certain alphavirus vectors to be intrinsically targeted for replication in dendritic cells and thus to elicit strong and comprehensive immune responses against multiple vaccine antigens (reviewed in Rayner, Dryga, and Kamrud, 2002, Rev. Med. Virol. 12:279-296). The alphavirus genus includes a diverse range of viruses that all belong to the Togaviridae family. Alphaviruses include Eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus (VEE), Everglades virus, Mucambo virus, Picnavirus, Western equine encephalitis virus (WEE), Sinbis virus, Semliki Forest virus, Middleburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kiziragachi virus, Highlands J virus, Fort Morgan virus, Ndom virus, and Buggy Creek virus. The viral genome is a single-stranded messenger sense RNA with a methylated cap at the 5' end and a variable length poly(A) sequence at the 3' end. The structural subunit, containing a single viral protein, C, resides within an icosahedral nucleocapsid associated with the RNA genome. In the virion, the capsid is surrounded by a lipid membrane and is covered with a regularly arranged series of transmembrane protein spikes, each of which usually consists of a heterodimeric complex of two glycoproteins, E1 and E2. See Pedersen et al., J. Virol 14:40 (1974). Sinbis virus and Semliki Forest virus are representative examples of alphaviruses and have been extensively studied.See Schlesinger, The Togaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986). VEE viruses have also been extensively studied. See, e.g., U.S. Patent No. 5,185,440 and other references cited therein.

[0007] Research into these viruses has led to the development of vaccines against alphavirus diseases and other diseases through the use of alphavirus vectors to deliver foreign DNA encoding antigens of interest. This is described in U.S. Patent No. 5,185,440 to Davis et al. and PCT Publication WO 92 / 10578. The introduction of exogenous, expressible DNA into eukaryotic cells is becoming a topic of increasing interest. It is well known that live attenuated virus vaccines are one of the most effective means of controlling viral diseases. However, for some viral (or other) pathogens, immunization with live virus strains may be impractical or unsafe. One alternative strategy is to insert sequences encoding immunogenic antigens of such agents into a viable, replicating strain of another virus. One such system utilizing a live VEE vector is described in U.S. Patent No. 5,505,947 to Johnston et al. Another such system is described by Hahn et al., 1992, Proc. Natl. Acad. Sci. USA 89:2679-2683, in which a Synbis virus construct expresses a truncated form of the influenza hemagglutinin protein. Another approach is to use infectious but growth-defective alphavirus particles. This is described in U.S. Patent No. 6,190,666 to Garoff et al., U.S. Patent Nos. 5,792,462 and 6,156,558 to Johnston et al., U.S. Published Patent Application No. 2002 / 0015945 A1 to Polo et al., U.S. Published Patent Application No. 2001 / 0016199 to Johnston et al., Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377, and Pushko et al. (1997) Virology 239:389-401. Alphaviruses have been shown to be relatively easy to genetically manipulate, which is reflected in several applications using alphaviruses as genomic expression libraries. See, e.g., U.S. Patent No. 6,197,502.The technique of expressing antigen libraries using Semiriki Forest virus (SFV) vectors has also been investigated in animal models. In this study, SFV particles expressing a library of tumor antigens were used to infect dendritic cells in vitro, and the dendritic cells were then used to immunize mice, demonstrating a certain degree of protection in a glioblastoma model (Yamanaka et al., 2001, J. Neurosurg. 94:474-81).

[0008] Several preclinical studies have demonstrated that fully allogeneic VRP vaccines enhance adaptive antigen-specific immunity and promote antitumor responses (de Mare A et al., Gene Ther. 2008;15(6):393-403; Lambeck AJ et al., Vaccine. 2010;28(26):4275-82; Riezebos-Brilman A et al., Gene Ther. 2007;14(24):1695-704). Due to their potential, various types of VRPs have been widely utilized and demonstrated to elicit antitumor immune responses against a variety of foreign and self-antigen targets expressed on cancer cells.

[0009] Preclinical studies have investigated the ability of VEE VRP vaccines to induce immune responses against various melanoma differentiation antigens (MDAs), including Tyr, gp100, and TRP-2. Among these vaccines, the VRP-TRP2 vaccine most potently activated TRP-2-specific cellular and humoral immunity and demonstrated potent preventive and therapeutic efficacy against B16 murine melanoma tumors (Avogadri et al., Plos One 2010;5(9):e12670). This study also demonstrated the importance of B cell responses to VRP cancer vaccines, highlighting the potential benefits of targeting tumor-associated antigens (TAAs). In this context, several studies have investigated the efficacy of targeting ErbB2 as a TAA, which is amplified or overexpressed in a subset of gastric and breast cancers. Vaccination with rat ErB2 (VRP-neu) encoding VEE VRP in combination with chemotherapy significantly delayed tumor progression in mice due to enhanced infiltration of antigen-specific T cells within tumors by VRP-neu vaccination (Eralp et al., Breast Cancer Res 2004;6(4):R275-R283). Subsequent studies investigated the efficacy of targeting neu using VRP-induced dendritic cells (VRP-DCs). These results showed that when tumor-bearing mice were inoculated with VRP-transduced dendritic cells (VRP-DCs), VRP-DCs expressing a truncated neu oncoprotein elicited neu-specific CD8 + VRP-DCs induced potent T cell and antibody responses. Furthermore, a single vaccination with VRP-DCs promoted the regression of large, established tumors in mice. This antitumor effect was mediated by CD4 + This result suggests that the VRP-DC vaccine-induced immune response against established tumors was completely abolished by T cell depletion. +These findings suggest that the efficacy of VRP vaccines is dependent on T cell and B cell responses (Moran et al., Vaccine 2007;25(36):6604-6612). Considering the possibility that the immunosuppressive microenvironment of various tumors may impair the induction of immune responses against TAAs, the efficacy of combining VRP vaccines with different immunomodulatory molecules has also been investigated. In vivo studies have shown that the immunogenicity and efficacy of VRP-TRP2 vaccines were improved when combined with immunomodulatory monoclonal antibodies (mAbs), such as CTLA-blocking antibodies or GITR-stimulating antibodies, by enhancing TRP-2-specific cellular and humoral immune responses (Avogadri et al., Cancer Immunol Res. 2014;2(5):448-458). In another preclinical study, a VRP-CEA vaccine was used to induce an immune response against carcinoembryonic antigen (CEA), which is often overexpressed in colorectal cancer. Tumor-bearing mice were vaccinated with a combination of VRP-CEA and VRP encoding IL-12 (VRP-IL12). This combined vaccination induced strong CEA-specific B and T cell responses and prolonged survival compared with a single injection of these vectors (Osada et al., Cancer Immunol Immunother. 2012;61(11):1941-51). Supporting the efficacy of IL-12, the therapeutic potential of SFV saRNA encoding IL-12 was demonstrated in another tumor model. Delivery of SFV-IL12 by electroporation enhanced tumor control and prolonged survival in tumor-bearing mice. Furthermore, the antitumor effect of electroporation of SFV-IL12 saRNA was enhanced by combination with a systemic PD-1 inhibitor (Silva-Pilipich et al., Mol Ther Nucleic Acids 2022;29:387-399).

[0010] IL-12 is a type 1 cytokine that has been investigated as a monotherapy for cancer. However, early clinical trials identified dose-limiting toxicities (e.g., Nguyen et al. (2020) Front. Immunol. 11:575-597). For example, "[i]n one phase II study, the maximum dose of 0.5 μg / kg / day caused severe side effects in 12 of 17 enrolled patients, resulting in two deaths," although the same dose was well tolerated in an earlier phase I study (ibid., citing Jenks (1996) J. Nat'l. Cancer Inst. 88:576-7). IL-12 has also been administered at lower doses, which were better tolerated but showed limited efficacy (ibid.). These studies generally utilized systemic (i.e., intravenous) or subcutaneous administration. However, one study examining these effects found infiltration of CD8+ T cells into metastatic lesions after treatment (Id.).

[0011] The tumor microenvironment is composed of various cell types, ranging from infiltrating immune cells to cancer cells, extracellular matrix, endothelial cells, and other cellular components and elements that influence tumor progression. This complex and intertwined balance not only differs between patients but also varies within lesions within the same patient (Jimenez-Sanchez et al. (2017) Cell 170(5):927-938). Tumor stratification based on tumor-infiltrating lymphocyte (TIL) and programmed death-ligand 1 (PD-L1) expression highlights the importance of the inflammatory environment for achieving objective responses to cancer (Teng et al. (2015) Cancer Res. 75(11):2139-45). A cross-cancer analysis of gene expression profiles from the Cancer Genome Atlas (TCGA) supports the correlation of tumor inflammatory signatures with objective responses to immunotherapy (Danaher et al. (2018) J. Immunother. Cancer 6(1):63).

[0012] In recent years, alternative vaccine administration routes, such as intravenous or intratumoral injection, have been evaluated in an attempt to improve cancer treatment by diversifying vaccine administration routes. For example, it has been shown that intravenous administration of an MVA vaccine encoding a heterologous antigen can induce a strong specific immune response against that antigen (see WO2014 / 037124). Furthermore, when CD40L was included in the MVA vaccine, the immune response was increased and enhanced.

[0013] Clearly, there is a substantial unmet medical need for additional cancer treatments, including active immunotherapies and cancer vaccines. Additionally, there is a need for therapies that can induce an enhanced immune response in multiple areas of a patient's immune response. In many aspects, embodiments of the present disclosure address these needs by providing vaccines and therapies that increase the immune response to tumors and improve currently available cancer treatments. Summary of the Invention

[0014] The present invention relates to a self-amplifying RNA (saRNA) for use in treating tumors, comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA increases the inflammatory response within the tumor, reduces the tumor growth rate and / or tumor size, and / or increases the overall survival of the subject, compared to non-intratumoral injection of the saRNA or injection of saRNA that does not contain a nucleic acid encoding IL-12, and the saRNA is administered intratumorally.

[0015] The present invention further provides methods of use and / or treatment involving one or more saRNAs of the present invention, in which one or more saRNAs of the present invention are administered intratumorally, intravenously, or intraperitoneally to a subject having a tumor. In some embodiments, the saRNAs of the present invention are used to prepare a medicament for increasing a subject's immune response to a tumor. In some embodiments, the saRNAs of the present invention are used to prepare a medicament for intratumoral injection, enhancing the subject's immune response to the injected tumor. In some embodiments, injection of the medicament into a tumor may reduce the size and / or growth rate of the injected tumor and may also reduce the size and / or growth rate of other tumors present in the subject that have not received intratumoral injection of the medicament (i.e., saRNA).

[0016] In some embodiments, the subject has a peritoneal tumor and the agent is used for intraperitoneal injection, thereby stimulating or enhancing an immune response against the peritoneal tumor.

[0017] The present invention provides saRNA for intravenous or intratumoral administration, comprising a nucleic acid encoding a TAA and a nucleic acid encoding IL-12, optionally in combination with CD40L. In some embodiments, the present invention provides saRNA for intratumoral and / or intravenous administration, comprising a nucleic acid encoding a TAA and IL-12. In other embodiments, the present invention provides a combination of saRNAs, one of which encodes IL-12 and at least one of which also encodes a TAA. This combination of saRNAs is administered to a subject and resides together in the subject's body for a period of time.

[0018] The features and advantages of various embodiments of the invention are described in more detail below. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Brief explanation of the drawings]

[0020] [Figure 1] Panels A, B, and C show the expression of the model antigen ovalbumin (OVA) in two different mouse cancer cell lines (4T1 and CT26) and a mouse fibroblast cell line (A31) at different time points after infection with VEEV-VRP-BFP-OVA (VRP-BN001) or SFV-VRP-BFP-OVA (VRP-BN010). Cells were seeded in 12-well plates and cultured overnight (O / N). The following day, they were either treated with a placebo (TNE buffer) or infected with VEEV-VRP-OVA-BFP or SFV-VRP-BFP-OVA at 30 TU / cell (cancer cells) or 10, 30, or 100 TU / cell (fibroblasts). After 1 hour, the medium was replaced with fresh cell culture medium containing 10% fetal calf serum (FCS). Every 24 hours, the cell culture medium was replaced with fresh medium (containing 10% FCS), and the collected cell supernatants were frozen and stored at -20°C for further analysis. This procedure was performed for 24, 48, 72, 96, and 120 hours after infection. After the last measurement point, the collected cell supernatants were used to measure the amount of OVA in the supernatants by ELISA. Panels A and B show the OVA concentrations (ng / ml) in the cell supernatants of 4T1 and CT26 cells infected with VRP-BN001 and VRP-BN010 at 30 TU / cell. Panel C shows the OVA concentrations (ng / ml) in the cell supernatants of A31 cells infected with VEEV-VRP-OVA-BFP and SFV-VRP-BFP-OVA at 10 TU / cell, 30 TU / cell, and 100 TU / cell. No OVA protein was detected in the placebo sample. Data are shown as mean ± standard error (SEM) (n = 3–6 per group). [Figure 2]Panels A and B show the effects of VEEV-VRP-OVA-BFP and SFV-VRP-BFP-OVA infection on the viability of different mouse cell lines. Cells were seeded into 96-well plates and cultured overnight. The following day, some cells were left untreated and maintained in fresh regular culture medium (+10% FCS). Other cells were treated with TNE buffer or infected with OVA and blue fluorescent protein (BFP) expressing 5–80 TU of VEEV-VRP and SFV-VRP per cell. After 1 hour, the medium was replaced with fresh cell culture medium containing 10% FCS. At 24 and 48 hours post-infection, CellTiter-Glo® 2.0 assay reagent was added to each well, and the plate was placed on an orbital shaker for 2 minutes to induce cell lysis. After incubation at room temperature (RT) for stabilization of the luminescence signal, luminescence was measured. Data were normalized to the control. A and B show the percentage viability of 4T1, CT26, and A31 cells 24 and 48 hours after infection with VEEV-VRP-OVA-BFP and SFV-VRP-BFP-OVA. Data are shown as mean ± standard error of the mean (SEM) (n = 4). [Figure 3] Panels A and B show ovalbumin (OVA) mRNA expression in tumors and tumor-draining lymph nodes (TdLNs) in 4T1 tumor-bearing mice 6 h after intratumoral (IT) administration of VEEV-VRP-OVA-BFP. 4T1 tumor cells were injected subcutaneously (SC) into the right flank of Balb / c mice. Seven days after tumor inoculation, mice were divided into groups (4–5 mice per group) and intratumorally administered 1×108 TU of VEEV-VRP-OVA-BFP or TNE buffer as a control. Six and 24 h after IT injection, mice were sacrificed, and organs were harvested, frozen, and subsequently used for RNA extraction and cDNA synthesis. qPCR was performed to measure the relative expression levels of OVA mRNA in different organs. HPRT was used as the reference gene. Panels A and B show the relative expression levels of OVA mRNA in mice 6 and 24 h after IT injection of TNE buffer or VEEV-VRP-OVA-BFP. Data are shown as mean ± standard error of the mean (SEM), n = 4–5 mice per group. [Figure 4]Panels A and B show the biodistribution of VEEV-VRP-OVA-BFP in tumors and tumor-draining lymph nodes (TdLNs) in B16.F10 tumor-bearing mice 6 hours after IT immunization. B16.F10 melanoma cells were injected subcutaneously (SC) into the right flank of C57BL / 6 mice. Seven days after tumor inoculation, mice were divided into groups (2-3 mice per group) and administered 1 x 108 TU of VEEV-VRP-OVA-BFP or TNE buffer as a control. Six hours after IT injection, mice were sacrificed, and tumors and TdLNs were harvested, pre-fixed in paraformaldehyde, and frozen. Seven-micrometer-thick tissue sections were prepared and stained. Images were captured using a fluorescence microscope. In A and B, representative images of sections stained with DAPI, FITC-conjugated CD45.2 antibody, BFP-conjugated unlabeled antiTagRFP antibody + AF647-conjugated secondary antibody, or a merged image are shown for tumors and TdLNs, respectively. White arrows indicate BFP+ cells on the images. [Figure 5] (A) IT injection of VRP-BN005 (VRP-Gp70) induces regression or complete rejection of CT26 colon cancer tumors. Balb / c mice were subcutaneously inoculated with 5 × 10 CT26.WT (wild-type) cells. 12 days later, mice were divided into groups and received IT injections of saline or 1 × 10 TU of VRP-Gp70. This day was designated as day 0. Mice received additional ("booster") IT immunizations on days 4 and 7 (vertical dotted lines). Tumor growth was monitored periodically. The number of cured mice is indicated in the lower right corner. (B) and (C) IT injection of VRP-BN005 (VRP-Gp70) induces peripheral antigen-specific CD8+ T cell responses. Mice were treated as described in Figure 5A. Two days after the final IT immunization, blood was collected and subjected to peptide restimulation. B shows the percentage of CD8+ T cells among surviving equivalent cells, and C shows the percentage of IFNγ+CD44+ T cells among CD8+ T cells in blood cells restimulated with AH-1 peptide. The dashed line indicates the development of tumor growth in animals with 1% or more IFNγ+CD44+ T cells among CD8+ T cells after restimulation with AH-1 peptide. Data are shown as mean ± standard error of the mean (SEM). n = 8 mice per group. [Figure 6](A) Repeated IT injections of VRP-BN005 (VRP-Gp70) induce transient tumor growth suppression in B16.F10 melanoma-bearing mice. C57BL / 6 mice were subcutaneously injected with 5 × 10 B16.F10 melanoma cells in the right flank. Seven days later, mice were divided into groups and received IT injections of saline or 1 × 10 TU of VRP-Gp70. This day was designated as day 0. Mice received additional ("booster") IT immunizations on days 5 and 8 (vertical dotted lines). Tumor growth was monitored periodically. (B) and (C) show that IT injections of VRP-BN005 (VRP-Gp70) induce peripheral antigen-specific CD8+ T cell responses. Mice were treated as described in (A). Four days after the final IT immunization, blood was collected and subjected to peptide restimulation. B shows the percentage of CD8+ T cells among surviving counterparts, and C shows the percentage of IFNγ+CD44+ cells among CD8+ T cells restimulated with p15E of the immunodominant antigen Gp70. Data are presented as mean ± standard error of the mean (SEM). n = 8 mice per group. [Figure 7] Figures A–C show that mice previously cured by IT treatment with VRP-BN005 (VRP-Gp70) reject tumors upon rechallenge. Naive Balb / c mice and Balb / c mice cured of CT26.WT tumors were rechallenged by subcutaneous injection of 5 × 10 CT26.WT tumor cells into the flank opposite the site of initial tumor placement. Tumor growth was measured periodically. Blood was collected 6 days before and 6 days after tumor rechallenge to examine antigen-specific CD8 T cell responses. Thirty-three days after tumor rechallenge, splenocytes were harvested and processed for the same purpose. All samples were used for restimulation with AH-1 peptide, and antigen-specific CD8+ cell responses were determined using flow cytometry. Figure A shows tumor-free survival of mice over time. Figure B shows the percentage of CD8+ T cells among surviving counterparts, and Figure C shows the percentage of IFNγ+CD44+ cells among CD8+ T cells. Data are expressed as mean ± standard error of the mean (SEM), n=5 mice per group. [Figure 8]Figures A–E show that IT injection of VRP-BN005 (VRP-Gp70) provided long-term protection against tumor rechallenge in mice by inducing antigen-specific CD8+ T cell memory responses. Naive and cured Balb / c mice were subcutaneously injected with CT26.WT as shown in Figures A–E and rechallenge. Thirty-three days after rechallenge, mice were sacrificed. Spleens, tumor-draining lymph nodes (TdLNs), and non-draining lymph nodes (non-dLNs) were collected from the injection sites of both naive and cured mice, and skin was collected from cured mice. Cells were isolated, stained, and analyzed by flow cytometry. Antigen-specific CD8+ T cells were identified by AH-1 pentamer staining. A shows the percentage of CD44+AH-1+ cells, B shows the percentage of CD127+CD62L+ central memory T cells (TCM), and C shows the percentage of CD127+CD62L- effector memory T cells (TCM) among viable CD8+ T cells in TdLN, non-dLN, spleen, and skin. D and E show the percentage of CD127+CD62L-CD69+CD103- and CD127+CD62L-CD69+CD103+ tissue-resident memory T cells (TRM) among viable CD8+ T cells in the skin of healed mice. Data are shown as mean ± standard error (SEM). n = 2-5 mice per group. [Figure 9]Figures A–D show that IT injection of VRP-BN005 (VRP-Gp70) induces CD4+ T cell memory responses. Naive and cured Balb / c mice were subcutaneously injected with CT26.WT as shown in Figure 8 and rechallenged. 33 days later, the mice were sacrificed. Spleens, tumor-draining lymph nodes (TdLNs), and non-draining lymph nodes (non-dLNs) were collected from the injection site of both naive and cured mice, and skin was collected from cured mice. Cells were isolated, stained, and analyzed by flow cytometry. Figure A shows the percentage of CD127+CD62L+ central memory T cells (TCMs). Figure B shows the percentage of CD127+CD62L- effector memory T cells (TEMs) among viable CD4+ T cells in TdLNs, non-dLNs, spleen, and skin. (C) and (D) Percentages of CD127+CD62L-CD69+CD103- and CD127+CD62L-CD69+CD103+ tissue-resident memory T cells (TRM) among viable CD4+ T cells in the skin of healed mice. Data are shown as mean ± standard error of the mean (SEM). n = 2–5 mice per group. [Figure 10] Panels A and B show the antitumor effect and changes in lymphocyte numbers / proportions in the tumor microenvironment (TME) of B16.F10 melanoma-bearing mice induced by a single intratumoral injection of VRP-BN005 (VRP-Gp70). C57BL / 6 mice were administered 5 × 10 B16.F10 cells subcutaneously in the right flank. Seven days later, when tumors measured >65 mm, mice were divided into groups and intratumorally injected with either TNE buffer or 1 × 10 TU of VRP-Gp70. Mice were sacrificed 1 and 7 days after immunization. Tumors were harvested, digested with collagenase / DNAse, and cells were stained and analyzed by flow cytometry. Panel A shows tumor weights on days 1 and 7. B shows the numbers of CD45+ cells, CD4+ T cells, CD8+ T cells, and NK cells (per mg of tumor) on days 1 and 7. Data are shown as mean ± standard error of the mean (SEM). n = 3-5 mice per group. [Figure 11A]This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. The percentages of CD69+, granzyme B+, or IFNγ+ NK cells are shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 11B] This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. At the same time points, cell supernatants were harvested and used for Luminex analysis to detect the secretion of multiple cytokines. The percentages of CD69+, granzyme B+, or IFNγ+ cells among CD8+ cells are shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 11C]This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. IL-12p70 concentrations (pg / ml) are shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 11D] This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. IFNγ concentrations (pg / ml) are shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 11E]This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. IL-10 concentrations (pg / ml) are shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 11F] This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. IL-6 concentrations (pg / ml) are shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 11G]This shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. The concentrations of GM-CSF (pg / ml) are indicated. Data are presented as mean ± standard error of the mean (SEM). [Figure 11H] This figure shows that IL-12-encoding VRP (VRP-IL12, VRP-BN006) induces immune cell activation more potently than VRP (VRP-BN015) in vitro. 1 × 10 splenocytes (derived from C57BL / 6 wild-type mice) were infected with VRP-BN015 or VRP-BN006 at 10, 30, or 100 TU / cell. Recombinant IL-12p70 (rIL12p70) was used at a final concentration of 200 ng / ml. Unstimulated cells were included as a negative control. After 18 hours, infected cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to assess CD8+ T cell and NK cell activation and cytotoxicity. Cell supernatants were harvested at the same time points and used for Luminex analysis to detect the secretion of multiple cytokines. The TNFα concentration (pg / ml) is shown. Data are presented as mean ± standard error of the mean (SEM). [Figure 12]Figures A–F show that repeated IT injections of VRP-BN006 (VRP-IL12) induce stronger antitumor effects and prolonged survival in B16.F10 tumor-bearing mice compared with VRP (VRP-BN015) injection, without causing IL-12-related cytotoxicity. C57BL / 6 mice were subcutaneously injected with 5 × 10 B16.F10 cells. Six days later, mice were divided into groups and administered 1 × 10 TU of VRP or increasing titers of VRP-IL12 (1 × 10 TU, 1 × 10 TU, or 1 × 10 TU) via IT injection. Control mice were treated IT with TNE buffer. This day was designated as day 0, and treatment was repeated on days 5 and 8 after the initial immunization (vertical dotted lines). Tumor growth was measured periodically. A-E show the mean tumor diameter (mm) in mice treated with TNE buffer, 1 x 10 TU VRP-BN015, 1 x 10 TU VRP-BN006, 1 x 10 TU VRP-BN006, and 1 x 10 TU VRP-BN006. F shows the survival rate of mice for all treatment groups. Data are shown as mean ± standard error of the mean (SEM). n = 5 mice per group. [Figure 13]Figures A–D show that repeated IT injections of a combination of VRP-BN005 (VRP-Gp70) and VRP-BN006 (VRP-IL12) induced enhanced antitumorigenic immune responses and improved survival in B16.F10 melanoma-bearing mice compared with IT injections of a combination of VRP-BN015 (VRP) and VRP-BN005 (VRP-Gp70). C57BL / 6 mice were subcutaneously injected with 5 × 10 B16.F10 cells. Eight days later, mice were divided into groups and immunized IT with either a combination of VRP-BN015 (VRP) + VRP-BN005 (VRP-Gp70) or a combination of VRP-BN005 (VRP-Gp70) + VRP-BN006 (VRP-IL12). The total VRP titer for these combinations was 1 × 10 TU. Control mice received TNE buffer IT injection. This day was designated as day 0. Booster inoculations were administered 5 days and thereafter after the initial immunization (vertical dotted lines). Tumor growth was measured periodically. (A-C) The mean tumor diameter (mm) is shown on days 0, 5, and 8 for mice treated with TNE buffer, a combination of VRP-BN015 (VRP) and VRP-BN005 (VRP-Gp70) (total 1 × 10 TU), and a combination of VRP-BN005 (VRP-Gp70) and VRP-BN006 (VRP-IL12) (total 1 × 10 TU). (D) Mouse survival rates for all treatment groups are shown. Data are presented as mean ± standard error (SEM). n = 5 mice per group. [Figure 14]Figures A–F show that repeated weekly IT injections of VRP-BN006 (VRP-IL12) can induce potent antitumor immune responses in B16.F10 tumor-bearing mice, similar to those induced by IT administration of VRP-IL12 (shorter intervals). C57BL / 6 mice were subcutaneously injected with 5 × 10 B16.F10 cells. Seven days later, mice were divided into groups and immunized IT with 1 × 10 TU of VRP or VRP-IL12. This day was designated as day 0. Booster inoculations of VRP and VRP-IL12 were repeated on days 5 and 8 or 7 and 14 after the initial immunization (vertical dotted lines). Control mice were treated IT with TNE buffer on days 0, 5, and 8. Tumor growth was measured periodically. A-C show mean tumor diameters (mm) on days 0, 5, and 8 for mice treated with TNE buffer, 1x108 VRP-BN015, or 1x108 TU VRP-BN006. D and E show mean tumor diameters (mm) on days 0, 7, and 14 for mice treated with 1x108 VRP-BN015 or 1x108 TU VRP-BN006. F shows mouse survival rates for all treatment groups. Data are shown as mean ± standard error of the mean (SEM). n=5 mice per group.

[0021] A brief description of arrays SEQ ID NO: 1 shows the BRP-OVA nucleotide sequence. SEQ ID NO: 2 shows the BFP-OVA amino acid sequence. SEQ ID NO: 3 shows the mouse GP70 nucleotide sequence. SEQ ID NO: 4 shows the mouse GP70 amino acid sequence. SEQ ID NO: 5 shows the mouse IL-12 nucleic acid sequence. SEQ ID NO: 6 shows the mouse IL-12 amino acid sequence. DETAILED DESCRIPTION OF THE INVENTION

[0022] The saRNAs and methods of the invention increase and enhance multiple aspects of a subject's immune response to one or more tumors. In various embodiments, the invention demonstrates that intratumor administration of saRNAs comprising a nucleic acid encoding at least one tumor-associated antigen (TAA) and a nucleic acid encoding IL-12 results in increased anti-tumor effects in a subject. As described in detail herein, these anti-tumor effects include, for example, reduced tumor size / volume, decreased tumor growth rate, increased overall survival, enhanced CD8+ T cell responses to the TAA, and enhanced inflammatory responses, including increased cytokine production, in the tumor, and in some embodiments, in the subject's systemic environment compared to administration of the saRNA alone.

[0023] In a further embodiment of the present invention, saRNA encoding IL-12, when administered in combination with saRNA encoding a TAA, further increases and / or enhances the immune response of a tumor-bearing subject and thus the effectiveness of the treatment.

[0024] As used herein, recombinant modified saRNA refers to saRNA containing at least one polynucleotide encoding a heterologous gene, such as a tumor-associated antigen (TAA). "Combination" refers to the simultaneous presence of one or more treatments in a subject. For example, a combination containing a saRNA encoding a TAA and a saRNA encoding IL-12 may be administered to a subject at the same time, even if they are administered to the subject at different times and / or by different routes of administration. Thus, the saRNAs in a combination treatment may be administered to a subject simultaneously, or at different times, as long as both are simultaneously present in the subject for a period of time (e.g., at least several hours, at least 12 hours, at least 24 hours, or at least two days or more). In some embodiments, IL-12 is encoded by the same saRNA. That is, in some embodiments, the saRNA of the present invention comprises a nucleic acid encoding a TAA and a nucleic acid encoding IL-12.

[0025] Thus, in some methods of the present invention, saRNAs encoding a TAA and IL-12 are injected intratumorally or intravenously into a tumor-bearing subject. In embodiments, the TAA and IL-12 are encoded by separate saRNAs, with at least one saRNA encoding at least one TAA, and in some embodiments, both saRNAs encode a TAA. In some embodiments, saRNAs encoding a TAA and IL-12 are administered to a subject to provide a combination of saRNA-encoded TAA and IL-12. That is, in some embodiments, IL-12 and a TAA are all encoded by the same saRNA, which can be administered to a subject to stimulate an immune response.

[0026] The present invention also provides saRNAs for preparing medicaments for intratumoral or intravenous injection for tumor treatment and / or for increasing an immune response in a subject against a tumor. In some embodiments, the medicament comprises saRNAs encoding a TAA and IL-12, optionally the same or different TAA. In some embodiments, the medicament comprises saRNAs encoding at least one TAA and IL-12, and in these embodiments, the nucleic acids encoding each of the TAA and IL-12 may be adjacent to each other in the saRNA, separated by nucleic acids encoding one or more other genes, or inserted at different positions in the saRNA.

[0027] In the examples provided herein, the inventors demonstrate that intratumorally administered saRNAs encoding tumor-associated antigens (TAA) and IL-12 increase and enhance a subject's immune response to the antigens. Thus, the present invention provides improved treatment for subjects with at least one tumor, including, for example, human cancer patients. More specifically, the inventors demonstrated that various saRNAs of the present invention and combinations thereof, when injected intratumorally, cause increased inflammation in the tumor. Observed indicators of systemic inflammation included increases in serum IL-12p70, M-CSF, and IL-33, increases in antigen-specific CD8+ T cells, increases in the percentage of CD8+ T cells expressing IFN-gamma and TNF-alpha, decreases in tumor size and / or growth rate, and improved survival rates.

[0028] Furthermore, data presented in the Examples herein demonstrated that subjects whose tumors were cured after treatment with a TAA and IL-12-encoding saRNA were more likely to reject the tumor when subsequently challenged with a newly transplanted tumor. Thus, the present invention provides compositions and methods of treatment that reduce the likelihood of tumor recurrence.

[0029] Thus, in one embodiment, the present invention includes a method for enhancing the immune response, reducing tumor size, and / or improving survival in a subject with a cancer tumor, the method comprising administering to the subject by intratumoral administration a recombinant, modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and IL-12, wherein the intratumoral administration of the saRNA increases and / or enhances the inflammatory response in the tumor, reduces tumor size, reduces tumor growth rate, and / or increases overall survival of the subject compared to the results expected from administration of the saRNA alone. In some embodiments, the method further comprises intratumorally administering to the subject a saRNA comprising a nucleic acid encoding a TAA that is the same as or different from a TAA encoded by another saRNA administered to the subject. In embodiments utilizing a combination of two or more saRNAs, the TAA can be encoded either by a saRNA that also encodes IL-12 or by a saRNA that encodes only the TAA.

[0030] In some embodiments, the present invention includes methods for increasing and / or enhancing the immune response, reducing tumor size, and / or increasing survival in a subject having a tumor, the methods comprising administering to the subject by intratumoral administration a recombinant, modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the intratumoral administration of the saRNA increases and / or enhances the inflammatory response in the tumor, reduces tumor size, reduces the tumor growth rate, and / or increases the overall survival of the subject compared to administration of a non-intratumoral saRNA virus comprising a first nucleic acid and a second nucleic acid encoding a TAA and IL-12, or intratumoral injection of the saRNA alone or non-intratumoral injection.

[0031] In additional embodiments, the present invention includes a method for enhancing an immune response, reducing tumor size, and / or increasing survival in a subject having a cancerous tumor, the method comprising administering to the subject intratumorally and / or intravenously a recombinant, modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein administration of the saRNA enhances the inflammatory response in the cancerous tumor, increases tumor reduction, and / or increases overall survival in the subject compared to injection of the saRNA alone or injection of saRNA comprising first and second nucleic acids encoding the TAA and IL-12 antigens administered by a different injection route (i.e., non-intratumoral or non-intravenous injection).

[0032] In some embodiments, the invention includes a method of enhancing an immune response, reducing tumor size, and / or increasing survival in a tumor-bearing subject, the method comprising administering to the subject by intratumoral administration a recombinant, modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein administration of the saRNA enhances T-cell responses specific to the TAA compared to intratumoral injection of the saRNA alone or non-intratumoral injection of a saRNA virus comprising a first nucleic acid and a second nucleic acid encoding the TAA and IL-12. In some embodiments, the TAA is encoded by a second saRNA that also encodes IL-12.

[0033] In some embodiments, the present invention includes methods for reducing tumor size and / or increasing survival in a subject with two or more tumors, the methods comprising administering, by intratumoral administration, to a particular tumor in the subject, a recombinant, modified saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein administration of the saRNA to the tumor reduces the growth rate and / or size of another tumor in the subject to which the saRNA(s) were not administered intratumorally. Thus, the present invention provides methods for stimulating an immune response against a tumor and / or reducing the size or growth rate of a tumor, comprising intratumoral injection of a different tumor.

[0034] In yet another embodiment, the present invention includes a method for inducing an enhanced inflammatory response in a cancerous tumor in a subject and / or throughout the subject's body, the method comprising intratumorally administering to the subject a recombinant, modified saRNA comprising a nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the intratumor administration of the saRNA results in an enhanced inflammatory response in the tumor compared to the inflammatory response produced or expected to be produced by non-intratumoral injection of a saRNA virus comprising the first and second nucleic acids encoding the heterologous tumor-associated antigen and IL-12.

[0035] In yet another embodiment, the present invention includes a method for inducing an increased and / or enhanced inflammatory response in a cancerous tumor in a subject, the method comprising intratumorally administering to the subject a recombinant, modified saRNA comprising a nucleic acid encoding a heterologous tumor-associated antigen (TAA) and IL-12, wherein administration of the saRNA results in an enhanced inflammatory response in the tumor compared to the inflammatory response produced by intratumoral or non-intratumoral injection of the saRNA alone, or by non-intratumoral injection of the saRNA comprising a nucleic acid encoding a heterologous tumor-associated antigen and IL-12.

[0036] In various further embodiments, the present invention provides a recombinant, modified saRNA for use in preparing a medicament for treating cancer or enhancing a subject's immune response to a cancerous tumor, wherein the saRNA comprises a nucleic acid encoding a tumor-associated antigen (TAA) and IL-12. Optionally, the saRNA further comprises a third nucleic acid encoding CD40L. Alternatively, the saRNA is provided in combination with a second saRNA comprising a nucleic acid encoding CD40L and, optionally, a TAA that is the same as or different from the TAA encoded by the first nucleic acid.

[0037] In various additional embodiments, the present invention includes a recombinant, modified saRNA used to enhance a subject's immune response to a tumor, the saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12.

[0038] In various additional embodiments, the present invention includes a recombinant, modified saRNA for use in preparing a medicament for treating cancer or enhancing an immune response in a subject with cancer, wherein the saRNA comprises a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12.

[0039] In embodiments in which two or more saRNAs are administered in a combination therapy, the saRNAs can be administered simultaneously or at different times, as long as they are present in the subject together for a certain period of time. In these embodiments, unless otherwise indicated, the saRNAs can be administered by the same route(s) and / or administration location, or by different locations and / or administration route(s). That is, in some embodiments, a first saRNA is administered intratumorally to a specific tumor in a subject, and a second or subsequent saRNA is administered intratumorally to a different tumor in the subject, or by intravenous, subcutaneous, intraperitoneal, or other administration route. In some embodiments, a first saRNA is administered intraperitoneally to a subject, and a second or subsequent saRNA is administered by a different administration route, for example, intravenous, subcutaneous, intratumoral, or other administration route.

[0040] In some embodiments, the TAA encoded by the at least one saRNA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 2 (TRP2), Brachyury, melanoma preferentially expressed antigen (PRAME), folate receptor 1 (FOLR1), human endogenous retrovirus-K envelope (HERV-K-env), human endogenous retrovirus-K-gag (HERV-K-gag), and combinations thereof. In some embodiments, the TAA encoded by the saRNA is expressed by at least one tumor to be treated, or is likely to be expressed or suspected to be expressed by at least one tumor to be treated.

[0041] The compositions and methods of the present invention enhance multiple aspects of a subject's immune response. In this way, the present invention provides improved treatment for subjects with at least one tumor, including, for example, cancer patients. More specifically, the inventors have demonstrated that various embodiments of the present invention, injected intratumorally, cause an increased inflammatory response in the tumor, which may also be detectable in the subject's serum. These indicators of systemic inflammation include increased production of IL-12p70, M-CSF, and IL-33; increased antigen-specific CD8+ T cells; increased percentage of CD8+ T cells expressing IFN-γ and TNF-α; decreased tumor size and / or growth rate; improved survival of treated subjects; and similar findings, which can be detected by assays known in the art, by evaluation of tumor and / or peripheral serum, periodic survival measurements, and similar techniques.

[0042] definition Before the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and does not limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0043] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "structural protein" includes one or more structural proteins, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or substitute for the methods described herein.

[0044] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0045] The term "about" when used in connection with a numerical value is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value, unless the context clearly indicates otherwise.

[0046] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option indicates that the first element is applicable without the second option. The second option indicates that the second element is applicable without the first option. The third option indicates that the first and second elements are applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. It is also understood that two or more options may be simultaneously applicable and therefore meet the requirements of the term "and / or."

[0047] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of other integers or steps, or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "comprising," or, occasionally, as used herein, with the term "having." Any of the above terms (comprising, containing, including, having) may, but are less preferred, replaced with the term "consisting of" whenever used herein in the context of an aspect or embodiment of the invention. As used herein, "consisting of" excludes elements, steps, or ingredients not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0048] A "mutated" or "modified" protein or antigen as described herein is defined herein as having any modification, such as deletion, addition, insertion and / or substitution, made to the nucleic acid or amino acid.

[0049] The term "antigen" includes all relevant epitopes of a particular compound, composition, or substance. The terms "epitope" or "antigenic determinant" refer to a site on an antigen to which B cells and / or T cells respond, alone or in combination with another protein (e.g., a major histocompatibility complex ("MHC") protein or a T cell receptor). Epitopes can be formed from contiguous amino acids or from noncontiguous amino acids juxtaposed by secondary and / or tertiary folding of a protein. Epitopes formed from contiguous amino acids typically are retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding typically are lost upon treatment with denaturing solvents. Epitopes typically comprise at least 5, 6, 7, 8, 9, 10, or more amino acids (usually fewer than 20 amino acids) in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0050] An antigen can be a tissue-specific (or tissue-associated) antigen or a disease-specific (or disease-associated) antigen. These terms are not mutually exclusive, as a tissue-specific antigen can also be a disease-specific antigen. A tissue-specific antigen is expressed in a limited number of tissues. An example of a tissue-specific antigen is prostate-specific antigen ("PSA"). A disease-specific antigen is expressed concurrently with the progression of a disease, and antigen expression correlates with or predicts the onset of a particular disease. An example of a disease-specific antigen is HER-2, which is associated with certain types of breast cancer, or PSA, which is associated with prostate cancer. A disease-specific antigen can be an antigen recognized by T cells or B cells.

[0051] Malignant growths arising from certain bodily tissues that have lost their characteristic structural differentiation are usually accompanied by increased cell division ability, invasion into surrounding tissues, and metastasis ability. Tumors can be benign or malignant. For example, prostate cancer is a malignant tumor that originates in or arises from prostate tissue, ovarian cancer is a malignant tumor that originates in or arises from ovarian tissue, colon cancer is a malignant tumor that originates in or arises from colon tissue, and lung cancer is a malignant tumor that originates in or arises from lung tissue. Residual cancer is cancer that remains in a subject after a treatment is administered to the subject to reduce or eradicate cancer. Metastatic cancer is cancer at one or more sites in the body other than the site of appearance of the original (primary) cancer from which the metastatic cancer originates.

[0052] A "conservative" variant is a variant protein or polypeptide having one or more amino acid substitutions that do not substantially affect or reduce the activity or antigenicity of the protein or its antigenic epitope. Generally, a conservative substitution replaces a particular amino acid with another amino acid having the same or similar chemical properties. For example, replacing a basic amino acid (e.g., lysine) with another basic amino acid (e.g., arginine or glutamine) is a conservative substitution. The term conservative variant also includes the use of a substituted amino acid in place of the unsubstituted parent amino acid, provided that antibodies generated against the substituted polypeptide also immunoreact with the unsubstituted polypeptide and / or the substituted polypeptide retains the function of the unsubstituted polypeptide. Non-conservative substitutions replace a particular amino acid with one having different chemical properties and typically reduce the activity or antigenicity of the protein or its antigenic epitope.

[0053] Specific non-limiting examples of conservative substitutions include the following: [Table 1]

[0054] A "disease-associated antigen" is expressed concurrently with a particular disease process, and antigen expression correlates with or predicts the onset of the disease. Disease-associated antigens include, for example, HER-2, which is associated with certain types of breast cancer, or prostate-specific antigen (PSA), which is associated with prostate cancer. Disease-associated antigens can be antigens recognized by T cells or B cells. Some disease-associated antigens may also be tissue-specific. Tissue-specific antigens are expressed in a limited number of tissues. Tissue-specific antigens include, for example, prostate-specific antigen (PSA).

[0055] The terms "tumor antigen" and "tumor-associated antigen" refer to antigens that are expressed only on, associated with, or overexpressed in tumor tissue. Examples of tumor antigens include, but are not limited to, 5-alpha-reductase, alpha-fetoprotein ("AFP"), AM-1, APC, April, B melanoma antigen gene ("BAGE"), beta-catenin, Bcl12, bcr-abl, Brachyury, CA-125, caspase-8 ("CASP-8," also known as "FLICE"), cathepsin, CD19, CD20, CD21 / complement receptor 2 ("CR2"), CD22 / BL-CAM, CD23 / F cεRII, CD33, CD35 / Complement receptor 1 (“CR1”), CD44 / PGP-1, CD45 / Leukocyte common antigen (“LCA”), CD46 / Membrane cofactor protein (“MCP”), CD52 / CAMPATH-1, CD55 / Decay accelerating factor (“DAF”), CD59 / Protectin, CDC27, CDK4, Carcinoembryonic antigen (“CEA”), c-myc, Cyclooxygenase-2 (“cox-2”), Deleted in Colorectal Cancer (“DCC”), DcR3, E6 / E7, CGFR, EMBP, Dna78, Farnesyltransferase fibroblast growth factor-8a ("FGF8a"), fibroblast growth factor-8b ("FGF8b"), FLK-1 / KDR, folate receptor, G250, G melanoma antigen gene family ("GAGE family"), gastrin 17, gastrin-releasing hormone, ganglioside 2 ("GD2") / ganglioside 3 ("GD3") / ganglioside monosialic acid-2 ("GM2"), gonadotropin-releasing hormone ("GnRH"), UDP-GlcNAc:R1Man(α1-6)R2[GlcNAc→Man(α1-6)]β1,6-N-acetylglucosaminyltransferase V (“GnT V”), GP1, gp100 / Pme117, gp-100-in4, gp15, gp75 / tyrosine-related protein-1 (“gp75 / TRP-1”), human chorionic gonadotropin (“hCG”), heparanase, Her2 / neu, human mammary tumor virus (“HMTV”), 70 kilodalton heat shock protein (“HSP70”), human telomerase reverse transcriptase (“hTERT”), insulin-like growth factor receptor-1 (“IGFR-1”), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-6), interleukin-11 (IL-6), interleukin-12 (IL-6), interleukin-13 (IL-6), interleukin-14 (IL-6), interleukin-15 (IL-6), interleukin-16 (IL-6), interleukin-17 (IL-6), interleukin-18 (IL-6), interleukin-19 (IL-6), interleukin-20 (IL-6), interleukin-21 (IL-6), interleukin-22 (IL-6), interleukin-23 (IL-6), interleukin-24 (IL-6), interleukin-25 (IL-6), interleukin-31 (IL-6), interleukin-16 (IL-6), interleukin-18 (IL-6), interleukin-19 (IL-6), interleukin-19 (IL-6), interleukin-19 (IL-6), interleukin-16 (IL-6), interleukin-19 (IL-6), IL-13 receptor (“IL-13R”), inducible nitric oxide synthase (“iNOS”), Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encoding family (“MAGE family”, e.g., at least MAGE-1, MAGE-2, MAGE-3, and MAGE-4), mammaglobin, MAP17, melanin A / melanoma antigen 1 recognized by T cells (“MART-1”), mesothelin, MIC A / B, MT-MMP, mucin, testis-specific antigen NY-ESO-1, osteonectin, p15, P170 / MDR1, p53, p97 / melanotransferrin, PAI-1, platelet-derived growth factor ("PDGF"), μPA, PRAME, probasin, progenipoietin, prostate-specific antigen ("PSA"), prostate-specific membrane antigen ("PSMA"), prostatic acid phosphatase ("PAP"), RAGE-1, Rb, RCAS1, SART-1, SSX family, STAT3, STn, TAG-72, transforming growth factor alpha ("TGF-α"), transforming growth factor beta ("TGF-β"), thymosin beta 15, tumor necrosis factor alpha ("TNF-α"), TP1, TRP-2, tyrosinase, vascular endothelial growth factor ("VEGF"), ZAG, p16INK4, and glutathione S-transferase ("GST").

[0056] "Adjuvant" refers to a vehicle for enhancing antigenicity. Adjuvants can include: (1) mineral (alum, aluminum hydroxide, and / or phosphoric acid) suspensions to which antigens are adsorbed; (2) water-in-oil emulsions in which antigen solutions are emulsified in mineral oil (Freund's incomplete adjuvant), optionally including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity by suppressing antigen degradation and / or inducing macrophage influx; (3) immunostimulatory substances (e.g., but not limited to, oligonucleotides, e.g., those containing CpG motifs) can also be used as adjuvants (see, e.g., U.S. Pat. Nos. 6,194,388 and 6,207,646); and (4) purified or recombinant proteins, such as costimulatory molecules (e.g., B7-1, ICAM-1, LFA-3, and GM-CSF).

[0057] As used herein, "affecting an immune response" includes the development in a subject of a humoral and / or cellular immune response to proteins and / or polypeptides produced by the saRNA or VRP of the present invention, and / or compositions and / or vaccines comprising the saRNA and VRP. A "humoral" immune response, as this term is known in the art, refers to an immune response that includes antibodies, while a "cellular" immune response, as this term is known in the art, refers to an immune response that includes T lymphocytes and other white blood cells, particularly an immunogen-specific response by HLA-restricted cytolytic T cells (i.e., "CTLs"). A cellular immune response occurs when a processed immunogen (i.e., peptide fragment) is presented in the context of a major histocompatibility complex.

[0058] As used herein, the term "alphavirus" has its conventional meaning in the art and includes the various species of Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Eastern equine encephalitis virus (EEEV). As used herein, "equine encephalitis virus (EEV)" includes VEEV, WEEV, and EEEV, as well as strains and isolates thereof.

[0059] The terms "alphavirus replicon particle," "virus replicon particle," "VRP," or "recombinant alphavirus particle," as used interchangeably herein, refer to a virion-like structural complex incorporating an alphavirus replicon RNA that expresses one or more heterologous RNA sequences. Typically, the virion-like structural complex comprises one or more alphavirus structural proteins embedded in a lipid envelope that encapsulates a nucleocapsid composed of the capsid and replicon RNA. The lipid envelope is typically derived from the plasma membrane of the cell in which the particle is produced. Preferably, the alphavirus replicon RNA is surrounded by a nucleoside structure composed of alphavirus capsid proteins, and the alphavirus glycoproteins are embedded in the cellular-derived lipid envelope. These replicon particles are propagation-deficient (or synonymously, "replication-deficient"), meaning that particles produced in a particular host cell are unable to produce progeny particles within that host cell due to a lack of helper function, i.e., alphavirus structural proteins necessary for packaging the replicon nucleic acid. However, the replicon nucleic acid can replicate itself and be expressed in a host cell into which it is introduced. The replicon particles of the present invention may be referred to as VEETC83 replicon particles, which refers to particles containing either TC83 replicon RNA or TC83 structural proteins, or both TC83 replicon RNA and TC83 structural proteins.

[0060] As used herein, the terms "expressed," "expressing," "expression," and the like, which can be used interchangeably, refer to transcription alone and both transcription and translation of a sequence of interest. Thus, when referring to the expression of a nucleotide sequence present in the form of DNA, the product resulting from this expression can be either RNA (resulting solely from transcription of the sequence to be expressed) or a polypeptide sequence (resulting from both transcription and translation of the sequence to be expressed). Thus, the term "expression" also includes the possibility that both RNA and polypeptide products result from the expression and remain together in the same shared environment. For example, this is the case when mRNA persists after being translated into a polypeptide product.

[0061] As used herein, an "expression cassette" is defined as a portion of a vector or recombinant virus typically used for cloning and / or transformation. An expression cassette typically consists of a) one or more coding sequences (e.g., an open reading frame (ORF), a nucleic acid encoding a gene, protein, and / or antigen), and b) a sequence (e.g., a promoter) that controls the expression of the one or more coding sequences. Additionally, an expression cassette may include a 3' untranslated region (e.g., a transcription terminator, e.g., a vaccinia transcription terminator). The term "expression cassette" can be used interchangeably with the term "transcription unit."

[0062] A "formulation" refers to a composition containing an active pharmaceutical or biological ingredient (e.g., a saRNA of the present invention) along with one or more additional ingredients. The term "formulation" is used interchangeably with the terms "pharmaceutical composition," "vaccine composition," and "vaccine formulation" herein. Formulations can be liquid or solid (e.g., lyophilized).

[0063] Additionally, vaccines can be monovalent or multivalent, and more than one cancer preventive nucleic acid molecule can be provided in the vaccine, either two or more of the selected nucleic acid molecules, or different nucleic acid molecules, including prime and booster vaccines that contain at least one nucleic acid molecule that is the same in both the prime and booster vaccines.

[0064] The term "gene" is used broadly to refer to any segment of a polynucleotide associated with a biological function. Thus, a gene may include introns and exons, such as in a genomic sequence, or only a coding sequence, such as a cDNA or viral RNA, and / or regulatory sequences required for expression. For example, a gene may also refer to a nucleic acid fragment that expresses mRNA or functional RNA or encodes a specific protein, including regulatory sequences.

[0065] The terms "nucleic acid," "nucleotide sequence," "nucleic acid sequence," and "polynucleotide" can be used interchangeably and refer to RNA or DNA that is linear or branched, single-stranded, double-stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides (e.g., methylated nucleotides and nucleotide analogs), uracil, other sugars, and linking groups (e.g., fluororibose and thiolate), and nucleotide branches. The sequence of nucleotides can be further modified after polymerization (e.g., by conjugation with a labeling component). Other types of modifications included in this definition are capping, substitution of one or more analogs of natural nucleotides, and introduction of a means for attaching a polynucleotide to a protein, metal ion, labeling component, other polynucleotides, or solid support. The polynucleotides may be obtained by chemical synthesis or may be derived from a microorganism.

[0066] As used herein, "operably linked" means that the components described are in a relationship that allows them to function in their intended manner (e.g., a promoter for transcribing a nucleic acid to be expressed). A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it is positioned so that it can direct transcription of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0067] "Percent (%) sequence homology or identity" with respect to the nucleic acid sequences described herein is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the reference sequence (i.e., the nucleic acid sequence from which it is derived) after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity, and does not take into account any conservative substitutions as part of the sequence identity. Alignment to determine percent nucleotide sequence identity or percent sequence homology can be performed in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment. This includes any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0068] For example, suitable alignment of nucleic acid sequences is provided by the following local homology algorithm: Smith and Waterman, (1981), Advances in Applied Mathematics 2:482-489. This algorithm can be applied to amino acid sequences by using a scoring matrix developed by: Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763. An exemplary implementation of this algorithm for determining percent identity of sequences in the "BestFit" utility application is provided by the Genetics Computer Group (Madison, Wis.). Default parameters for this method are described in the Wisconsin Sequence Analysis Package Programs Manual, Version 8 (1995) (available from the Genetics Computer Group, Madison, Wis.). In the context of the present invention, a preferred method for determining percent identity is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages, the Smith-Waterman algorithm can be used, using default parameters for the scoring table (e.g., gap open penalty: 12, gap extension penalty: 1, gap: 6). From the data generated, the "match" value reflects "sequence identity." The same applies mutatis mutandis to "percent (%) amino acid identity."Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art; for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort = HIGH SCORE; database = non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR. Details of these programs can be found at the following internet address: http: / / http: / / blast.ncbi.nlm.nih.gov / .

[0069] The terms "pharmaceutical product," "pharmaceutical composition," and "medicament" are used interchangeably herein and refer to a substance and / or combination of substances used for the prevention or treatment of disease.

[0070] By "pharmaceutically acceptable" it is meant that the carrier or excipient, at the dosages and concentrations employed, does not cause undesirable or adverse effect(s) in the subject(s) to which it is administered.

[0071] "Pharmaceutically acceptable carriers" are described, for example, in Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975); Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis

[2000] ; and Handbook of Pharmaceutical Excipients, 3rd Edition, A. Kibbe, Ed., Pharmaceutical Press (2000). These describe compositions and formulations using conventional pharmaceutically acceptable carriers suitable for administering the vectors and compositions disclosed herein. Generally, the nature of the carrier used will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like) as a vehicle. For solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, pH buffering agents, and the like, for example, sodium acetate or sorbitan monolaurate).

[0072] As used herein, "preventing," "preventing," "prevention," or "prevention" of a disease or infection means preventing such disease from occurring in a subject (e.g., a human or animal).

[0073] The term "prime-boost vaccination" refers to a vaccination strategy that uses an initial priming injection of a vaccine targeting a specific antigen, followed by one or more boost injections of the same vaccine at intervals. Prime-boost vaccination can be homologous or heterologous. Homologous prime-boost vaccination uses a vaccine containing the same immunogen and vector for both the priming injection and one or more boost injections. Heterologous prime-boost vaccination uses a vaccine containing the same immunogen for both the priming injection and one or more boost injections, but uses different vectors for the priming injection and one or more boost injections. For example, homologous prime-boost vaccination may use a saRNA vector containing the same nucleic acid expressing an alphavirus antigen for both the priming injection and one or more boost injections. In contrast, heterologous prime-boost vaccination may use a saRNA vector containing a nucleic acid expressing one alphavirus protein for the priming injection and another saRNA vector expressing a second alphavirus protein not included in the priming injection, or vice versa. Heterologous prime-boost vaccination also encompasses various combinations (e.g., using a plasmid encoding an immunogen in the priming injection and using saRNA encoding the same immunogen in one or more boosting injections, or using a recombinant protein immunogen in the priming injection and using a saRNA vector encoding the same protein immunogen in one or more boosting injections).

[0074] As used herein, the term "promoter" refers to a regulatory region of nucleic acid (usually DNA) located upstream of the nucleic acid sequence to be expressed. This region contains specific DNA sequence elements that are recognized and bound by protein transcription factors and polymerases responsible for synthesizing RNA from the coding region of the promoted gene, for example. Because promoters are usually located immediately adjacent to the gene in question, a position within the promoter is designated relative to the transcription start site where transcription of DNA begins for a particular gene (i.e., an upstream position is a negative number counted backward from -1; e.g., -100 is a position 100 base pairs upstream). Thus, a promoter sequence may include nucleotides up to position -1. However, it should be noted that nucleotides from position +1 are not part of the promoter; i.e., in this regard, the translation initiation codon (ATG or AUG) is not part of the promoter. Thus, SEQ ID NO: 7 or 8 is a polynucleotide comprising a promoter of the present invention. The term "26S promoter" is well known to those skilled in the art and refers to the subgenomic promoter of the 26S RNA of an alphavirus, which is typically contained in a single open reading frame (e.g., capsid E3-E2-6K-E1 of VEEV). The mRNAs encoding the structural proteins of EEV (e.g., VEEV) are typically transcribed from replication intermediates and the 26S subgenomic RNA promoter.

[0075] Advantageously, nucleic acids encoding the structural proteins of alphaviruses, i.e., capsid, E1 glycoprotein, and E2 glycoprotein, contain at least one attenuating mutation. As used herein, the terms "attenuating mutation" and "attenuating amino acid" refer to a nucleotide mutation or encoded amino acid that allows for such a mutation to result in a reduced probability of causing disease in the host (i.e., loss of virulence), whether the mutation is a substitution mutation or an in-frame deletion or addition mutation, in accordance with standard terminology in the art (see, e.g., B. Davis, et al. Microbiology 132 (3d ed. 1980)). The phrase "attenuating mutation" excludes mutations that may be lethal to the virus unless such mutations are used in combination with a "restoring" mutation that renders the virus viable despite being attenuated. In certain embodiments, the helper nucleic acid(s) comprise at least one attenuating mutation.

[0076] The terms "protein," "peptide," "polypeptide," and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. A polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass amino acid polymers that are modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as a label or conjugation with a biologically active moiety).

[0077] The term "recombinant" when applied to nucleic acids and vectors refers to nucleic acids, vectors, or those created by or containing the artificial combination of two or more heterologous nucleic acid sequence segments. Artificial combinations are most commonly achieved by the artificial manipulation of isolated segments of nucleic acids using established genetic engineering techniques. Generally, "recombinant" saRNAs described herein refer to saRNAs produced using standard genetic engineering methods; i.e., the saRNAs of the present invention are genetically engineered or genetically modified saRNAs. Thus, the term "saRNA" includes saRNAs (e.g., VRPs) that have a recombinant nucleic acid stably integrated into a genome, preferably in the form of a transcription unit. The transcription unit may include a promoter, enhancer, terminator, and / or silencer. The saRNAs of the present invention can express a heterologous antigenic determinant, polypeptide, or protein (antigen) upon induction of regulatory elements.

[0078] The term "reference sample" as used herein refers to a sample that is analyzed in substantially the same manner as the sample of interest, and its information is compared with the information of the sample of interest.Therefore, the reference sample provides a standard for evaluating the information obtained from the sample of interest.The reference sample can be identical to the sample of interest, except for one component that can be replaced, missing, or added.

[0079] The term "structural protein" of EEV refers to the structural protein / polyprotein encoded by the RNA of EEV (e.g., any of WEEV, VEEV, or EEEV described herein). Structural proteins are typically produced by the virus as a structural polyprotein of five proteins (i.e., C, E3, E2, 6k, and E1), commonly represented in the literature as C-E3-E2-6k-E1. E3 and 6k are also described as membrane translocation / transport signals for two glycoproteins (E2 and E1). As used herein, a nucleotide sequence encoding a "structural protein" refers to a nucleotide sequence encoding proteins required for encapsidation (e.g., packaging) of the viral genome, including the capsid protein, E1 glycoprotein, and E2 glycoprotein. The "structural polyprotein" of EEV refers to the EEV polyprotein C-E3-E2-6k-E1.

[0080] As used herein, the term "transcription level" or "protein level" with respect to a particular promoter refers to the amount of a gene / nucleic acid product present in a body or sample at a given time. Transcription or protein level (e.g., transcription of a nucleic acid as mRNA or the amount of protein translated from mRNA) can be determined, measured, or quantified by the mRNA or protein expressed from a gene / polynucleotide, for example, encoded by the saRNA of the present invention. Gene expression can result in the production of a protein by transcription of the gene by RNA polymerase to produce messenger RNA (mRNA) containing the same protein-coding information, and translation of the mRNA by ribosomes to produce the protein. The term "transcribed" or "transcription" refers to the process of copying the DNA sequence of a gene into mRNA by RNA polymerase using DNA as a template. The term "translated" or "translation" refers to the process in which the information contained in mRNA is used as a blueprint to synthesize a protein. Transcription or protein levels can be quantified, for example, by normalizing the amount of mRNA or protein of interest present in a sample with the total amount of gene products (mRNA or total protein) of the same category in the same sample or a reference sample (e.g., collected at the same time from the same sample). Transcription can be measured or detected by any method known in the art (e.g., methods for direct detection and measurement of the gene product of interest, which typically act through binding of the gene product of interest to one or more distinct molecules) or by detection means specific for the gene product of interest (e.g., primer(s), probe, antibody, protein scaffold). Such methods include, for example, RT-PCR and / or quantitative PCR. Protein levels can be measured or detected by any known method known to those skilled in the art (e.g., Western blot, ELISA, or mass spectrometry).

[0081] As used herein, "treat," "treating," or "treatment" of a disease refers to preventing, reducing, ameliorating, partially or completely alleviating, or curing a disease (e.g., a disease caused by EEV), which may be one or more of reducing the severity of the disease, limiting or preventing the onset of symptoms characteristic of the disease being treated, inhibiting the worsening of symptoms characteristic of the disease being treated, limiting or preventing the recurrence of the disease in a subject previously affected by the disease, and limiting or preventing the recurrence of symptoms in a subject.

[0082] As used herein, "trivalent" in conjunction with a vaccine or saRNA means that the vaccine or saRNA is valenced against three different viruses and generates a protective immune response against antigens (e.g., structural proteins or structural polyproteins) of those different viruses. Thus, in the context of the trivalent saRNA vaccine of the present invention, trivalent refers to a valence against three different viruses, where the antigen is encoded in the saRNA vaccine or vaccine containing saRNA expressing a nucleic acid encoding the antigen (e.g., structural proteins or structural polyproteins of VEEV, WEEV, and EEEV). Another example of trivalency that is also included in the meaning of trivalent is when the three different viruses are different viral strains (e.g., two WEEV strains, e.g., 71V-1658 and Fleming, in addition to VEEV or EEEV strains). In the latter case, the saRNA of the present invention includes nucleotide sequences encoding proteins (e.g., structural proteins, structural polyproteins, envelope proteins) of, for example, WEEV 71V-1658, WEEV Fleming, and EEEV strains (e.g., EEEV V105-00210). In comparison, "monovalent" means that the vaccine or saRNA has a valency against only one virus of a particular species (e.g., only VEEV, only WEEV, or only EEEV) and generates a protective immune response against only one structural protein or structural polyprotein of a single virus. However, it does not exclude the generation of a protective immune response against several closely related viral subtypes. Thus, "bivalent" means that the vaccine or saRNA has a valency against two viruses.

[0083] "Vector" refers to a recombinant DNA or RNA plasmid or virus containing a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide may contain a sequence of interest for prophylactic or therapeutic purposes and may optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replicating in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0084] The term "viral replicon" as used in the context of the present invention refers to an RNA or DNA comprising a portion of the 49S viral genomic RNA that is essential for transcription and cytoplasmic amplification of the transported RNA and for subgenomic RNA expression of a heterologous nucleic acid sequence. Thus, the replicon encodes and expresses viral nonstructural proteins necessary for cytoplasmic amplification of the viral RNA.

[0085] In the context of the present invention, the term "virus" or "recombinant virus" refers to an infectious or non-infectious virus comprising a viral genome. In this case, the nucleic acids, promoters, recombinant proteins, and / or expression cassettes referred to herein are part of the viral genome of the respective recombinant virus. The recombinant viral genome is packaged, and the resulting recombinant virus can be used to infect cells and cell lines, and in particular to infect live animals, including humans.

[0086] The term "TCID50" is an abbreviation for "tissue culture infectious dose," and the amount of a pathogen that produces pathological changes in 50% of inoculated cell cultures is the TCID 50 Expressed in TCID / ml. 50 Methods for determining this are well known to those skilled in the art and are described, for example, in Example 2 of WO03 / 053463.

[0087] As used herein, the term "subject" refers to a living multi-cellular vertebrate organism (e.g., a human, a non-human mammal, or a (non-human) primate). As used herein, the term "subject" may be used interchangeably with the term "animal."

[0088] As used herein, increased survival rate can be characterized as the increased survival rate of subjects (for example, human cancer patients), but can also be characterized in terms of clinical trial endpoints that are understood in the art.Some exemplary clinical trial endpoints associated with increased survival rate include, but are not limited to, overall survival (OS), progression-free survival (PFS), etc.

[0089] Combinations and Methods In various embodiments, the present invention includes a saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, which, when administered intratumorally, induces both an enhanced inflammatory response and an enhanced T cell response compared to the inflammatory response and T cell response induced by non-intratumor administration of the saRNA alone or by non-intratumor administration of a saRNA comprising a first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12.

[0090] Increased or enhanced inflammatory response In various aspects of the present disclosure, administration of the saRNA of the present invention has been shown to induce an increased or enhanced inflammatory response compared to administration of the saRNA alone. This increased or enhanced inflammatory response can be detected, for example, by measuring cytokine levels in the subject's blood and / or plasma, or can be detected at or near the site of administration, for example, in tumors injected intratumorally. Thus, in one embodiment of the present invention, intratumoral administration of the saRNA of the present invention has been shown to induce an increased or enhanced inflammatory response in tumors compared to administration of the saRNA alone.

[0091] In some embodiments, saRNA encoding a tumor-associated antigen (TAA) and encoding IL-12 is injected intraperitoneally to treat a subject and induce an increased or enhanced inflammatory response in at least one peritoneal tumor and / or the omentum.

[0092] In some embodiments, the subject treated by the method of the present invention has at least one tumor that is peritoneal dissemination, or has malignant ascites or omental metastatic tumor, preferably derived from abdominal malignant tumor, more preferably derived from ovarian cancer or colon cancer. In some embodiments, the subject is being treated for a tumor that is preferably an abdominal malignant tumor that metastasizes to the peritoneal cavity and / or omentum. In some embodiments, the subject has a tumor that is an ovarian cancer or colon cancer tumor.

[0093] In some embodiments, treating a subject with a method of the invention increases the subject's chances of survival, hi some embodiments, treating a subject with a method of the invention induces an antigen-specific immune or T cell response, or IFN-γ production in the subject's peritoneal cavity and / or omentum.

[0094] In some embodiments, intraperitoneal administration is performed in a prime-boost regimen.

[0095] In another embodiment, the present invention provides a pharmaceutical preparation or composition comprising the saRNA of the present invention, wherein the pharmaceutical preparation or composition is adapted for intraperitoneal administration.

[0096] In yet another embodiment, the present invention provides a saRNA of the present invention for use in improving the overall survival of a subject, preferably a human, suffering from peritoneal dissemination or malignant ascites or omental metastatic tumor, preferably from an abdominal malignancy, more preferably from ovarian cancer or colon cancer, wherein the saRNA is administered intraperitoneally.

[0097] In yet another embodiment, the present invention provides a saRNA of the present invention for use in alleviating signs or symptoms of peritoneal dissemination, malignant ascites, or omental metastatic tumor in a subject, preferably a human, in some embodiments, where the tumor originates from an abdominal malignancy, e.g., ovarian cancer or colon cancer, and the saRNA is administered intraperitoneally.

[0098] In yet another embodiment, the present invention provides a saRNA of the present invention for use in eliciting an antigen-specific immune response or T cell response or inducing interferon-γ (IFN-γ) production in the peritoneal cavity of a subject suffering from peritoneal dissemination, malignant ascites, or a metastatic tumor of the omentum, e.g., one resulting from an abdominal malignancy such as ovarian cancer or colon cancer, wherein the saRNA is administered intraperitoneally.

[0099] In at least one embodiment, an "increased inflammatory response" or "enhanced inflammatory response" according to the present disclosure is characterized by one or more of the following: increased production of IL-12p70, M-CSF, and / or IL-33; increased antigen-specific CD8+ T cells; increased percentage of CD8+ T cells expressing IFN-gamma and TNF-alpha; decreased tumor size and / or growth rate; improved survival of treated subjects; and the like, as may be detected by assays known in the art. As used herein, an "increased inflammatory response" generally refers to an increase in the production of a particular cytokine or cell type associated with inflammation compared to baseline levels prior to treatment with the methods and / or compositions of the invention. For example, an "increased inflammatory response" may involve at least a 10%, 20%, 30%, 50%, 70%, or 100% or greater increase in the amount of a cytokine or cell type compared to baseline levels prior to treatment with the methods and / or compositions of the invention.

[0100] As used herein, "enhanced inflammatory response" generally refers to an inflammatory response in which new cytokines or new cell populations are produced that were undetectable or only detectable in small amounts prior to treatment with the methods and / or compositions of the invention.

[0101] The compositions and methods of the present invention enhance multiple aspects of a subject's immune response. In this way, the present invention provides improved treatment for subjects with at least one tumor, including, for example, cancer patients. More specifically, the inventors have demonstrated that various saRNAs of the present invention and combinations thereof, when injected intratumorally, resulted in an increased inflammatory response in tumors that was detectable within the tumor and also detectable in the subject's serum. These indicators of systemic inflammation include increased production of IL-12p70, M-CSF, and IL-33; increased antigen-specific CD8+ T cells; increased proportions of CD8+ T cells expressing IFN-γ and TNF-α; decreased tumor size and / or growth rate; improved survival of treated subjects; and similar findings, which can be detected by assays known in the art, such as tumor and / or peripheral serum assessments, periodic survival measurements, and similar techniques.

[0102] Thus, in accordance with the present disclosure, an enhanced or increased inflammatory response in a tumor and / or tumor cells can be determined by measuring increased expression of one or more molecules indicative of an increased inflammatory response, including secretion of chemokines and cytokines, as known in the art. Exemplary inflammatory response markers include one or more of IL-12p70, M-CSF, IL-33, IFN-gamma, and TNF-alpha. These molecules and their measurement are well-established assays understood in the art and can be performed according to known techniques. See, e.g., Borrego et al. ((1999) Immunology 7(1):159-165).

[0103] The increased or enhanced inflammatory response provided by the compositions and methods of the invention may also result in a decrease in the volume and / or mean diameter of at least one tumor in a treated subject. In this manner, the invention provides a method of reducing the volume, size, and / or growth rate of at least one tumor in a subject. In some embodiments, treatment with the compositions and / or methods of the invention results in a decrease in the volume, size, and / or growth rate of at least one tumor by at least 10%, 20%, 30%, 50%, or more compared to the volume, size, and / or growth rate of the tumor before treatment.

[0104] Enhanced T cell responses According to the present application, "enhancing T cell responses" refers to (1) CD8 + Increased frequency of T cells, (2) CD8 + Increased T cell activation, and (3) CD8 + Therefore, in accordance with the present application, whether or not a T cell response is enhanced is characterized by one or more of: (1) increased proliferation of CD8 + Increased frequency of T cells, (2) CD8 + Increased T cell activation, and / or (3) CD8 + This can be determined by measuring the expression of one or more molecules that indicate increased T cell proliferation. + Exemplary markers useful for measuring T cell frequency, activation, and proliferation include IFN-γ, TNF-α, and / or CD44, as are known in the art. The frequency of antigen-specific T cells can also be measured using MHC multimers, such as pentamers or dextramers. Such measurements and assays, as well as others suitable for use in evaluating the methods and compositions of the invention, are validated and understood in the art.

[0105] In one embodiment, CD8 + Increased frequency of T cells was observed in IFN-γ and / or dextramer-positive CD8 cells compared to pretreatment / baseline values. +Characterized by at least a two-fold, three-fold, five-fold, or even ten-fold increase in the number of CD8 T cells. + Increased T cell activation can be achieved by, for example, CD8 + Characterized as at least a two-fold increase in the number of T cells and / or at least a two-fold increase in CD69 and / or CD44 expression compared to pre-treatment / baseline expression. + Increased T cell proliferation is characterized, for example, by at least a two-fold increase in Ki67 expression compared to pre-treatment / baseline expression.

[0106] In an alternative embodiment, the increase or enhancement of the T cell response is achieved by CD8 + The treatment is characterized by increased T cell effector cytokine expression and / or increased cytotoxic effector function. Increased effector cytokine expression can be measured, for example, by expression of one or more of IFN-γ, TNF-α, and / or IL-2 compared to pre-treatment / baseline. Increased cytotoxic effector function can be measured, for example, by expression of one or more of CD107a, granzyme B, and / or perforin, and / or antigen-specific killing of target cells. The assays, cytokines, markers, and molecules described herein, as well as their measurement, are established and understood in the art and can be performed according to known techniques. Further cytokine measurements and assays for measuring T cell responses can be found in the Examples.

[0107] In yet further embodiments, the combinations and methods described herein are used to treat human cancer patients. In preferred embodiments, the cancer patient has and / or has been diagnosed with a cancer selected from the group consisting of breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, gastric cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, cervical cancer, or colorectal cancer. In yet further embodiments, the combinations and methods described herein are used to treat human cancer patients having and / or having been diagnosed with breast cancer, colorectal cancer, or melanoma.

[0108] Tumor-Associated Antigens for Use in the Compositions and Methods of the Invention In certain embodiments, the immune response is generated in the subject against a cell-associated polypeptide antigen. In certain such embodiments, the cell-associated polypeptide antigen is a tumor-associated antigen (TAA). In various embodiments, the TAA is HER2, PSA, PAP, CEA, MUC-1, survivin, TRP1, TRP2, Brachyury, melanoma-preferentially expressed antigen (PRAME), folate receptor 1 (FOLR1), human endogenous retrovirus-K envelope (HERV-K-env), or human endogenous retrovirus-K-gag (HERV-K-gag), either alone or in any combination thereof.

[0109] In further embodiments, the TAA is selected from the group consisting of 5 alpha reductase, alpha fetoprotein, AM-1, APC, April, BAGE, beta-catenin, Bcl12, bcr-abl, CA-125, CASP-8 / FLICE, cathepsin, CD19, CD20, CD21, CD23, CD22, CD33, CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6 / E7, CGFR, EMBP, Dna78, farnesyltransferase, FGF8b, FGF8 a, FLK-1 / KDR, folate receptor, G250, GAGE ​​family, gastrin-17, gastrin-releasing hormone, GD2 / GD3 / GM2, GnRH, GnTV, GP1, gp100 / Pmel17, gp-100-in4, gp15, gp75 / TRP1, hCG, heparinase, Her2 / neu, HMTV, Hsp70, hTERT, IGFR1, IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, MAGE family, mammaglobin, MAP17, melan-A / MART-1, mesothelin, MIC These may include, but are not limited to, A / B, MT-MMP, mucin, NY-ESO-1, osteonectin, p15, p170 / MDR1, p53, p97 / melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin, progenipoietin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta, thymosin-beta-15, TNF-alpha, TRP1, TRP2, tyrosinase, VEGF, ZAG, p16INK4, and glutathione-S-transferase.

[0110] In some embodiments, the TAA is an endogenous retroviral protein (ERV) or a derivative thereof. Such an ERV can be an ERV belonging to the human HERV-K protein family, such as a HERV-K envelope (env) protein, a HERV-K group-specific antigen (gag) protein, and a HERV-K "melanoma risk marker" (mel) protein (see, e.g., Cegolon et al. (2013) BMC Cancer 13:4).

[0111] Any TAA may be used as long as at least one objective or desired result of the present invention (e.g., stimulation of an immune response) is achieved after administration of the saRNA containing the TAA. In some embodiments, the TAA encoded by one or more saRNAs is known to be expressed in at least one tumor of the subject, for example, based on previous testing of tumor samples. Exemplary sequences of TAAs, including those referred to herein, are known in the art and suitable for use in the compositions and methods of the present invention. The sequences of the TAA used in the compositions and methods of the present invention may be identical to sequences known in the art or disclosed herein, or may have less than 100% sequence identity (e.g., at least 90%, 91%, 92%, 95%, 97%, 98%, 99% or more) with any of the nucleotide or amino acid sequences known in the art or disclosed herein. That is, the sequence of a TAA used in a composition or method of the invention can differ by fewer than 20 nucleotides or amino acids, or by fewer than 19, 18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide or amino acid from a reference sequence known in the art and / or disclosed herein, provided that at least one objective or desired result of the invention (e.g., helping to stimulate an immune response when administered to a subject as a component of a saRNA) is achieved. Those of skill in the art are familiar with techniques and assays for evaluating a TAA to ensure its suitability for use in a saRNA or method of the invention.

[0112] In certain embodiments, modifications to one or more TAAs, such as, but not limited to, HERV-K env, HERV-K gag, HERV-K mel, CEA, MUC-1, PAP, PSA, PRAME, FOLR1, HER2, survivin, TRP1, TRP2, or Brachyury, are made so that polyclonal antibodies primarily reactive with one or more of the TAAs described herein are elicited after administration to a subject. Such antibodies can attack and eliminate tumor cells and prevent metastatic cells from developing and leading to metastasis. The effector mechanism for this anti-tumor effect is mediated by complement-dependent and antibody-dependent cellular cytotoxicity (ADCC). In addition, the induced antibodies can suppress cancer cell proliferation through growth factor-dependent oligodimerization and inhibition of receptor internalization. In certain embodiments, such modified TAAs can induce CTL responses against known and / or putative TAA epitopes presented by tumor cells.

[0113] In certain embodiments, the modified TAA polypeptide antigen comprises a CTL epitope and a variant of a cell-associated polypeptide antigen, wherein the variant comprises at least one CTL epitope or a foreign TH epitope. Certain such modified TAAs can, in one non-limiting example, comprise one or more HER2 polypeptide antigens comprising at least one CTL epitope and a variant comprising at least one CTL epitope of a foreign TH epitope; these HER2 antigens and methods for making them are described in U.S. Patent No. 7,005,498 and U.S. Patent Application Publication Nos. 2004 / 0141958 and 2006 / 0008465, which are incorporated herein by reference.

[0114] IL-12 Structurally, IL-12 is a type I cytokine heterodimeric protein consisting of two covalently linked p35 and p40 subunits. The heterodimeric form is also called IL-12-p70 or IL-12-p35 / p40. IL-12 has many effects in promoting immune responses, but several clinical trials using IL-12 have resulted in unacceptable levels of adverse events (Lasek et al. (2014) Cancer Immunol. Immunother. 63: 419-35). IL-12 induces the production of IFN-gamma, which promotes T cell proliferation. H It has been demonstrated that IL-12 induces differentiation of IL-1 cells and also increases the activation and cytotoxicity of T cells and NK cells (Nguyen et al. (2020) Front. Immunol. 11:575-597). Various modified forms of IL-12 are known in the art and are useful in embodiments of the present invention as long as they retain IL-12 functions, such as increasing the secretion of IFN-gamma ("IFN-γ"). For example, a modified form of IL-12 known in the art is "single-chain interleukin-12," also referred to as "IL-12sc" or "scIL-12." This IL-12sc offers the advantage of automatically having the correct stoichiometry of the p35 and p40 subunits, so that excess p40 subunits, which can exert an inhibitory effect on full-length IL-12, are not produced (see, e.g., Anderson et al. (1997) Hum. Gene Ther. 8:1125-35). Homodimers of p40 subunits have been shown to inhibit the activity of the heterodimeric forms and are therefore not useful in embodiments of the present invention.

[0115] In some embodiments of the present invention, IL-12 is encoded by a saRNA together with a tumor-associated antigen ("TAA"). In some embodiments, the saRNA encodes IL-12 and, optionally, the TAA. In some embodiments, the IL-12 sequence is a murine IL-12 sequence. In some embodiments, the IL-12 has an amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:6, or has an amino acid sequence that differs by less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from the amino acid sequence set forth in SEQ ID NO:6, or is identical to the sequence set forth in SEQ ID NO:6. In additional embodiments, the nucleic acid encoding IL-12 comprises a nucleic acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:5, i.e., differs by less than 20, 10, 5, 4, 3, 2, or 1 nucleic acid in the sequence from the nucleic acid sequence set forth in SEQ ID NO:5, or is identical to the sequence set forth in SEQ ID NO:5. Because IL-12 has been well studied, one of skill in the art would be able to introduce sequence modifications into more variable or less conserved regions without affecting gene function. Any IL-12 sequence is considered suitable for use in embodiments of the present invention, as long as it provides at least one function of IL-12 in an assay, such as any of the assays for IL-12 used in the Examples or otherwise known in the art.

[0116] Self-amplifying RNA Currently, there are two different types of synthetic RNA vaccines: conventional mRNA and self-amplifying RNA (saRNA). The use of conventional mRNA strategies (also called non-replicating or non-amplifying mRNA) against infectious diseases and cancer has been investigated in several preclinical and clinical trials. In vitro-transcribed mRNAs encoding viral antigens have been investigated as vaccines, while those encoding therapeutic proteins (e.g., antibodies or immunomodulatory drugs) are considered immunotherapies. Incorporation of chemically modified nucleotides, sequence optimization, and various purification strategies improve mRNA translation efficiency and reduce inherent immunogenic properties. However, antigen expression is proportional to the number of conventional mRNA transcripts efficiently delivered during vaccination. Therefore, achieving adequate expression for protection or immunomodulation may require large doses or repeated administrations. saRNA vaccines, which are genetically engineered replicons derived from self-replicating single-stranded RNA viruses, address this limitation. They can be delivered as viral replicon particles (VRPs), in which the saRNA is packaged into viral particles, or as fully synthetic saRNA generated after in vitro transcription. To generate replication-defective VRPs, envelope proteins are provided in trans as defective helper constructs during production. Therefore, the resulting VRPs lack the ability to form infectious viral particles after initial infection, and only the RNA is capable of further amplification. VRPs can be derived from both positive-strand and negative-strand RNA viruses, but the latter are more complex and require reverse genetics to rescue the VRPs. Similar to gene therapy, there are several issues associated with the use of viral vectors for vaccine development. These include the immunogenicity of the vector itself, which can elicit unwanted immune responses and prevent subsequent booster doses using the same vector. Pre-existing immunity to the viral vector can also render the vaccine ineffective. Like live-attenuated vaccines, replication-competent alphavirus vectors pose the threat of viral reactivation. To circumvent this, saRNA vaccines can be produced and administered in a similar manner to conventional mRNA vaccines.Positive-strand alphavirus genomes commonly used in saRNA vaccine design include Venezuelan equine encephalitis virus (VEE), Sindbis virus (SINV), and Semliki Forest virus (SFV). Alphavirus replicase genes encode RNA-dependent RNA polymerase (RdRP) complexes that amplify synthetic transcripts in situ. Antigen or therapeutic sequences are expressed at high levels as separate, distinct entities, eliminating the need for further proteolytic processing of the immunogen. As a result of their self-replicating activity, saRNA can be delivered at lower concentrations than conventional mRNA vaccines to achieve comparable antigen expression.

[0117] As mentioned above, saRNA constructs have historically been derived from alphaviruses (e.g., Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), or Sindbis virus). These saRNA constructs contain four nonstructural proteins, a subgenomic promoter, and a gene of interest (which replaces the viral structural proteins). By deleting the viral structural proteins, the RNA cannot generate infectious virus. After delivery to the cytoplasm, the nonstructural proteins form an RNA-dependent RNA polymerase (RDRP), which replicates both the genomic RNA (the entire RNA strand) and the subgenomic RNA (the gene of interest). Each of the four nonstructural proteins participates in the formation of the RDRP, a complex, multistep process. This RNA replication results in higher antigen expression than non-replicating mRNA.

[0118] EEV virus, protein, and nucleotide sequences EEV is an alphavirus belonging to the Togaviridae family. It is a small, enveloped, positive-strand RNA virus well known in the art. The viral nucleocapsid is surrounded by a host-derived lipid membrane in which a trimer of E1 and E2 heterodimeric envelope proteins is embedded. The nucleocapsid consists of a capsid protein (C) surrounding a single-stranded RNA genome. The EEV viral RNA genome (49S RNA) is approximately 11-12 kb in length, contains a 5' cap and a 3' polyadenylated tail, and is translated immediately upon entry into the cell. The 5' region of the genome encodes four nonstructural proteins (NSP1, NSP2, NSP3, and NSP4). The 3' region of the genome encodes five structural proteins (C, E3, E2, 6k, and E1) that are expressed as a structural polyprotein from the 26S subgenomic RNA. mRNAs encoding the structural proteins are transcribed from replication intermediates and the 26S subgenomic promoter. Proteolytic cleavage of the polyprotein generates the mature structural proteins C, E3, E2, 6k, and E1. The nucleocapsid (C) protein has autoproteolytic activity, which cleaves the C protein from the precursor protein immediately after the ribosome passes through the junction between the C and E3 protein-coding sequences. Subsequently, the envelope glycoproteins E2 and E1 are derived by proteolytic cleavage to form a heterodimer. E2 initially appears in infected cells as a precursor pE2 consisting of E3 and E2. After glycosylation and passage through the endoplasmic reticulum and Golgi apparatus, E3 is cleaved from E2 by a furin-like protease activity at the cleavage site.

[0119] A live attenuated vaccine is used by the US military and laboratory personnel, and a formalin-inactivated vaccine is available for horses.

[0120] One such live attenuated vaccine is TC-83, originally developed by the U.S. Army for use in vaccines (Pittman et al., 1996). TC-83 was generated by serial passage of the Trinidad donkey VEEV strain in guinea pig heart cells (Alevizatos et al., 1967). Point mutations in the E2 and 5' untranslated regions contribute to the attenuated phenotype of TC-83 (Kinney et al., 1993). TC-83 is known to be effective in preventing human disease; however, 15–37.5% of vaccine recipients develop fever symptoms (Berge et al., 1961; McKinney et al., 1963; Alevizatos et al., 1967; Pittman et al., 1996), and only 82% of vaccine recipients seroconvert upon vaccination. The probability of maintaining plaque-reducing neutralizing titers of 1:20 or greater over 5–8 years was 60%. Because TC-83 is available only as an investigational vaccine and in a limited population, additional studies to evaluate the vaccine's immunogenicity in humans over time are unavailable. Of interest to this study is that after intranasal infection with the vaccine strain of VEEV-TC-83, C57BL / 6 (WT) mice developed disseminated brain infection with high infectious titers without mortality (Hart et al., 1997; Steele et al., 1998; Julander et al., 2008; Taylor et al., 2012).

[0121] It should be understood that any combination of any of the above-mentioned WEEVs, EEEVs, and / or VEEVs is encompassed by any of the embodiments described herein.

[0122] As used herein, "adjuvant effect" refers to the enhancement of the immune response generated by a particular protein or component encoded by the saRNA, which is induced by other encoded proteins or components of the saRNA.

[0123] In some embodiments, the compositions, methods, and combinations of the present invention increase overall survival of treated subjects. As used herein, "increased overall survival" intends a statistically significant improvement in survival rates of treated subjects compared to untreated subjects.

[0124] Expression cassette / control sequence In various embodiments, one or more nucleic acids described herein are incorporated into one or more expression cassettes in which the one or more nucleic acids are operably linked to expression control sequences. "Operably linked" or "operably linked" means that the described components are in a relationship that allows them to function in their intended manner, e.g., a promoter that allows transcription and expression of the nucleic acid. An expression control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. Expression control sequences include, but are not limited to, an appropriate promoter, enhancer, transcription terminator, a start codon at the beginning of the open reading frame encoding the protein, splicing signals for introns, and in-frame stop codons. Suitable promoters include, but are not limited to, the SV40 early promoter, the RSV promoter, retroviral LTR, the adenovirus major late promoter, the human CMV immediate-early I promoter, and various poxvirus promoters (including, but not limited to, promoters derived from vaccinia virus or MVA and promoters derived from FPV, such as the 30K promoter, the I3 promoter, the PrS promoter, the PrS5E promoter, the Pr7.5K promoter, the PrHyb promoter, the Pr13.5 long promoter, the 40K promoter, the MVA-40K promoter, the FPV40K promoter, the 30K promoter, the PrSynIIm promoter, the PrLE1 promoter, and the PR1238 promoter). Additional promoters are further described in WO2010 / 060632, WO2010 / 102822, WO2013 / 189611, WO2014 / 063832 and WO2017 / 021776, which are incorporated by reference herein in their entireties.

[0125] Additional expression control sequences include, but are not limited to, leader sequences, stop codons, polyadenylation signals, and any other sequences required for the proper transcription and subsequent translation of the nucleic acid sequence encoding the desired heterologous protein (e.g., TAA and / or IL-12) in the desired host system. The vector may also contain additional elements required for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the desired host system. Those skilled in the art will further appreciate that such vectors are readily constructed using conventional methods (Ausubel et al. (1987) in "Current Protocols in Molecular Biology," John Wiley and Sons, New York, NY) and are commercially available.

[0126] Methods and Dosing Regimen for Administering the Combination In one or more aspects, the combinations of the present invention can be administered as part of an allogeneic and / or xenogeneic prime-boost regimen. As shown in the Examples, allogeneic prime-boost regimens increase specific T cell responses in a subject. Thus, in one or more embodiments, there are combinations and / or methods for stimulating an immune response, reducing tumor size, and / or increasing survival in a subject, comprising administering to the subject a combination of the present invention, wherein the combination is administered as part of an allogeneic or xenogeneic prime-boost regimen.

[0127] Methods for the generation of saRNA Conventional and synthetic saRNA vaccines are generated in essentially the same way. Briefly, a DNA-dependent RNA polymerase promoter (usually derived from T7, T3, or SP6 bacteriophage) and an mRNA expression plasmid (pDNA) encoding the RNA vaccine candidate are engineered as a template for in vitro transcription. The flexibility of the gene synthesis platform is a key advantage. In conventional mRNA vaccines, the antigen or immune regulatory sequence is flanked by 5' and 3' untranslated regions (UTRs). A poly(A) tail can be incorporated from the 3' end of the pDNA template or added enzymatically after in vitro transcription. The saRNA vaccine pDNA template contains additional alphavirus replicon genes and conserved sequence elements. Nonstructural proteins 1, 2, 3, and 4 (nsP1-4) form the RdRP complex and are therefore essential for replicon activity. In vitro transcription is typically performed on a linearized pDNA template or linear DNA fragment, usually using T7 DNA-dependent RNA polymerase, resulting in multiple copies of the RNA transcript. For efficient translation, the 5' end is capped. This can usually be done by co-transcriptional capping with a synthetic cap analog or post-transcriptional enzymatic capping. After the RNA is 5'-capped and purified, it is ready for formulation and delivery. In the case of saRNA, the co-transcriptional capping step with a cap analog is usually preferred because the 5' cap differs from that of conventional mRNA.

[0128] The RNA product is then purified, which may include removing in vitro transcription by-products in the form of double-stranded dsRNA. For example, these can be removed by chromatography using the double-strand-specific enzyme RNase or a substance with specific dsRNA affinity. Additional chromatography or other purification steps (e.g., affinity purification, filtration) can be used to enhance the purity and quality of the RNA product. Affinity purification may also include a polyA-specific resin to enrich for full-length and polyadenylated RNA and remove short by-products.

[0129] vaccine In certain embodiments, the saRNA of the present disclosure can be formulated as part of a vaccine or used to prepare a pharmaceutical product that is a vaccine. When preparing a vaccine, the saRNA can be converted into a physiologically acceptable form.

[0130] An exemplary preparation method is as follows: 5 x 10 purified virus 8 TCID 50 The vaccine is stored at -80°C in a solution of 10 mM Tris, 140 mM NaCl (pH 7.4) at a titer of 1 x 10 / ml. 8 ~1×10 9The virus particles can be lyophilized in ampoules, preferably glass ampoules, in phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin. Alternatively, vaccine doses or shots can be prepared by stepwise lyophilizing the virus in the formulation. In certain embodiments, the formulation contains additional additives, such as mannitol, dextran, sugars, glycine, lactose, polyvinylpyrrolidone, and optional other additives, such as antioxidants, inert gases, stabilizers, or recombinant proteins (e.g., human serum albumin) suitable for in vivo administration. The ampoules can then be sealed and stored at a suitable temperature, e.g., 4°C to room temperature, for several months. However, for long-term storage, the ampoules are preferably stored at temperatures below -20°C, most preferably at about -80°C.

[0131] In various embodiments involving vaccination or therapy, the lyophilizate is dissolved in 0.1 to 0.5 ml of aqueous solution, preferably saline or a Tris buffer such as 10 mM Tris, 140 mM NaCl (pH 7.7). The saRNA vaccine or pharmaceutical composition of the present disclosure can be dissolved in 10 ml of solution. 4 ~10 10 TCID 50 / ml, 10 5 ~5×10 9 TCID 50 / ml, 10 6 ~5×10 9 TCID 50 / ml or 10 7 ~5×10 9 TCID 50 It is believed that the compound can be formulated to a concentration range of 10 / ml. 6 ~10 10 TCID 50 Contains 10 6 TCID 50 , 10 7 TCID 50 , 10 8 TCID 50 , 5×10 8 TCID 50 , 10 9TCID 50 , 5×10 9 TCID 50 or 10 10 TCID 50 Optimization of the dosage and number of doses administered is within the ability and knowledge of one skilled in the art.

[0132] The dosage in humans and animals is approximately 1 x 10 4 ~Approx. 1×10 10 and advantageously about 1 x 10 per dose 6 ~Approx. 1×10 8 In a vaccine-based immunogenic approach, the inventors contemplate weekly, biweekly, or monthly dosing for a period of about 1 month to about 12 months or longer. Booster vaccinations can be administered thereafter as needed, e.g., annually.

[0133] In one or more preferred embodiments, the saRNA, as defined herein, is administered intratumorally to the cancer patient. In other embodiments, the saRNA is administered intraperitoneally to the cancer patient. In other embodiments, the saRNA is administered to the cancer patient intratumorally, intravenously, subcutaneously, and / or intraperitoneally, either simultaneously or at different times.

[0134] Kits, Compositions, and Methods of Use In various embodiments, the invention includes kits, pharmaceutical combinations, pharmaceutical compositions, and / or immunogenic combinations comprising one or more saRNAs comprising the nucleic acids described herein.

[0135] It is contemplated that kits and / or compositions of the invention may include one or more containers or vials of one or more recombinant poxviruses of the present disclosure, along with instructions for administering the saRNA(s). In more specific embodiments, it is contemplated that the kits may include instructions for administering an initial priming dose of the saRNA(s), followed by one or more additional boost doses of the saRNA(s), if appropriate, in a homologous or heterologous prime-boost regimen.

[0136] The kits and / or compositions provided herein may generally include one or more pharmaceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, diluents, and / or stabilizers. Such auxiliary substances may include water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or similar substances. Suitable carriers are typically large, slowly metabolized molecules (e.g., proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and similar substances).

[0137] Embodiment Embodiment 1 is a saRNA for use in stimulating an immune response to a tumor-associated antigen (TAA) in a subject, comprising: (a) a first nucleic acid encoding the tumor-associated antigen (TAA); and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA increases and / or enhances the inflammatory response in the tumor, decreases the growth rate and / or size of the tumor, and / or increases the overall survival of the subject, compared to non-intratumoral administration of the saRNA or injection of a saRNA that does not contain a nucleic acid encoding IL-12, and wherein the saRNA is administered intratumorally.

[0138] Embodiment 2 is the saRNA for use according to embodiment 1, further comprising (c) a third nucleic acid encoding 4-1BBL.

[0139] Embodiment 3 is a saRNA for use as described in embodiment 1, wherein said TAA is an endogenous retroviral (ERV) protein.

[0140] Embodiment 4 is the saRNA for use according to embodiment 1, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, and combinations thereof.

[0141] Embodiment 5 is a saRNA for use in treating a tumor, comprising (a) a first nucleic acid encoding a tumor-associated antigen (TAA), and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA enhances the inflammatory response in the tumor, reduces the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or injection of a saRNA that does not contain a nucleic acid encoding IL-12, and wherein the saRNA is administered intratumorally.

[0142] Embodiment 6 is the saRNA for use according to embodiment 5, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 2 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, and combinations thereof.

[0143] Embodiment 7 is a pharmaceutical combination comprising: (i) a saRNA comprising (a) a first nucleic acid encoding a tumor-associated antigen (TAA) and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA enhances the inflammatory response in a tumor, reduces the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or injection of a saRNA that does not contain a nucleic acid encoding IL-12; and (ii) a pharmaceutically acceptable carrier.

[0144] Embodiment 8 is a method for inhibiting tumor growth and / or increasing survival in a subject having a cancerous tumor, the method comprising intratumorally administering to the subject an saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA), a second nucleic acid encoding IL-12, and optionally a third nucleic acid encoding 4-1BBL, wherein the intratumor administration of the saRNA enhances the inflammatory response in the tumor, reduces tumor growth and / or size, and / or increases overall survival of the subject compared to injection of an saRNA that does not include a nucleic acid encoding IL-12 or an saRNA alone.

[0145] Embodiment 9 is the method of embodiment 8, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), Brachyury, PRAME, FOLR1, HERV-K-env, HERV-K-gag, and combinations thereof.

[0146] Embodiment 10 is the method of embodiment 8, wherein the subject is a human cancer patient.

[0147] Embodiment 11 is a method of reducing tumor size or growth and / or increasing survival in a tumor-bearing subject, the method comprising intraperitoneally administering to the subject a saRNA comprising a first nucleic acid encoding IL-12 and, optionally, a second nucleic acid encoding a tumor-associated antigen (TAA), wherein the administration of the saRNA enhances natural killer (NK) cell responses and enhances CD8 T cell responses specific for the TAA compared to a baseline value before treatment or compared to an expected result from administering the saRNA alone.

[0148] Embodiment 12 is the method of embodiment 11, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), Brachyury, PRAME, FOLR1, HERV-K-env, HERV-K-gag, and combinations thereof.

[0149] Embodiment 13 is the method of embodiment 11, wherein the subject is a human and the tumor is in the abdominal cavity.

[0150] Embodiment 14 is a method for generating an enhanced inflammatory response in an intraperitoneal tumor in a subject, the method comprising intraperitoneally administering to the subject an saRNA comprising a first nucleic acid encoding IL-12 or IL-12sc and, optionally, a second nucleic acid encoding a heterologous tumor-associated antigen (TAA), wherein the intraperitoneal administration of the saRNA generates an enhanced inflammatory response in the tumor compared to the inflammatory response that would result from non-intraperitoneal administration of the saRNA alone.

[0151] Embodiment 15 is the method of embodiment 14, further comprising intraperitoneally administering to the subject a booster dose of the same saRNA.

[0152] Embodiment 16 is a vaccine comprising any of embodiments 1-6 and a pharmaceutically acceptable carrier.

[0153] Embodiment 17 is a combination of the saRNA of any one of embodiments 1 to 6, the vaccine of embodiment 16, or the pharmaceutical product of embodiment 7, for use in reducing tumor size and / or increasing survival in a subject with a cancerous tumor.

[0154] Embodiment 18 is a combination of the saRNA described in any one of Embodiments 1 to 6, the vaccine described in Embodiment 16, or the pharmaceutical product described in Embodiment 7, used in a method for reducing tumor size or increasing survival in a subject having a cancerous tumor, the method comprising administering the saRNA, vaccine, or pharmaceutical product combination intratumorally or intraperitoneally to the subject, wherein the intratumor or intraperitoneal administration enhances the inflammatory response in the cancerous tumor, reduces the tumor growth rate, promotes tumor shrinkage, and / or increases overall survival of the subject, compared to injection of the saRNA alone.

[0155] Embodiment 19 is a combination of the saRNA described in any one of embodiments 1 to 6, the vaccine described in embodiment 16, or the pharmaceutical described in embodiment 7, used in a method for stimulating an immune response in a subject, the method comprising intratumorally or intraperitoneally administering the saRNA, vaccine, or pharmaceutical combination to the subject, wherein the intratumor or intraperitoneal administration enhances an inflammatory response in the tumor, detectable by analysis of the tumor or the subject's blood or serum, compared to administration of the saRNA alone, or compared to non-intratumor or non-intraperitoneal administration of the saRNA, or compared to intratumor or intraperitoneal administration of a saRNA lacking one or more of the components encoded by the saRNA.

[0156] Embodiment 20 is a combination of the saRNA described in any one of embodiments 1 to 6, the vaccine described in embodiment 16, or the pharmaceutical described in embodiment 7 for use in a method for treating cancer in a subject.

[0157] Embodiment 21 is a combination of the saRNA of any one of embodiments 1 to 6, the vaccine of embodiment 16, or the pharmaceutical product of embodiment 7 for use in a method for treating cancer, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, head and neck cancer, thyroid cancer, melanoma, gastric cancer, bladder cancer, kidney cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, ovarian cancer, urothelial, cervical or colorectal cancer.

[0158] Embodiment 22 is the saRNA of any one of embodiments 1 to 6, wherein the enhanced inflammatory response is localized to the tumor.

[0159] Embodiment 23 is a method for inducing an enhanced inflammatory response in an intraperitoneal tumor in a subject, the method comprising intratumorally administering to the subject an saRNA comprising a first nucleic acid encoding a first heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12 or IL-12sc, wherein the intratumoral administration of the saRNA produces an enhanced inflammatory response in the tumor compared to the inflammatory response produced by or that would result from intratumoral injection of an saRNA virus alone.

[0160] Embodiment 24 is the method of embodiment 23, wherein the saRNA further comprises a nucleic acid encoding 4-1BBL.

[0161] Embodiment 25 is a saRNA comprising (a) a first nucleic acid encoding a tumor-associated antigen (TAA) and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA enhances the inflammatory response in the tumor, reduces the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or injection of a saRNA that does not contain a nucleic acid encoding IL-12.

[0162] Embodiment 26 is the saRNA of embodiment 25, further comprising (c) a third nucleic acid encoding 4-1BBL.

[0163] Embodiment 27 is the saRNA of embodiment 25, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, p15, and combinations thereof.

[0164] Embodiment 28 is a pharmaceutical combination comprising the saRNA of embodiment 25 and a pharmaceutically acceptable carrier.

[0165] Embodiment 29 is a method of stimulating an immune response in a subject having multiple tumors, comprising administering locally (intratumorally) to some, but not all, of the tumors in the subject an saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12, thereby stimulating an immune response in the subject against the TAA.

[0166] Embodiment 30 is a method of treating a subject having at least one inaccessible tumor and at least one accessible tumor, comprising administering locally (intratumorally) to at least one accessible tumor of the subject an saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding 4-1-BBL, thereby reducing or stopping the growth of the inaccessible tumor.

[0167] Embodiment 31 is a method for preventing or reducing the extent of tumor recurrence or metastasis in a subject having at least one tumor, comprising administering intratumorally or intraperitoneally to at least one tumor in the subject an saRNA comprising at least one first nucleic acid encoding IL-12 and optionally a second nucleic acid encoding a TAA, thereby reducing or stopping the growth of the inaccessible tumor.

[0168] Embodiment 32 is the method of embodiment 29, 31, or 31, wherein the saRNA further comprises a nucleic acid encoding 4-1BBL.

[0169] Embodiment 33 is a saRNA used to stimulate an immune response to a tumor-associated antigen (TAA) in a subject, comprising (a) a first nucleic acid encoding IL-12, e.g., scIL-12, and (b) a second nucleic acid encoding the TAA, wherein intraperitoneal administration of the saRNA enhances or increases the inflammatory response in a tumor, decreases the growth rate and / or size of the tumor, and / or increases the overall survival of the subject, compared to non-intraperitoneal administration of the saRNA or administration of a saRNA that does not include a nucleic acid encoding IL-12, and wherein the saRNA is administered intraperitoneally.

[0170] Embodiment 34 is a saRNA for use as described in embodiment 33, further comprising (c) a third nucleic acid encoding 4-1BBL.

[0171] Embodiment 35 is the saRNA for use according to embodiment 33, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, and combinations thereof.

[0172] Embodiment 36 is a saRNA for use in treating tumors, comprising (a) a first nucleic acid encoding a tumor-associated antigen (TAA), and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA enhances / or increases the inflammatory response in the tumor, reduces the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or administration of a saRNA that does not contain a nucleic acid encoding IL-12, and wherein the saRNA is administered intratumorally.

[0173] Embodiment 37 is the saRNA for use according to embodiment 36, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 2 (TRP2), Brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, and combinations thereof.

[0174] Embodiment 38 is a pharmaceutical combination comprising: (i) a saRNA comprising (a) a first nucleic acid encoding a tumor-associated antigen (TAA) and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA enhances and / or increases the inflammatory response in the tumor, decreases the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or administration of a saRNA that does not comprise a nucleic acid encoding IL-12; and (ii) a pharmaceutically acceptable carrier.

[0175] Embodiment 39 is a saRNA used to stimulate an immune response to a tumor-associated antigen (TAA) in a subject, the saRNA comprising a nucleic acid encoding IL-12, wherein intraperitoneal administration of the saRNA increases the inflammatory response in a tumor, optionally a peritoneal tumor, and / or the omentum, decreases the growth rate and / or size of the tumor, and / or increases the overall survival of the subject, compared to non-intraperitoneal administration of the saRNA or administration of a saRNA that does not contain a nucleic acid encoding IL-12, and the saRNA is administered intraperitoneally.

[0176] Exemplary embodiments of the invention will now be described in detail, examples of which are illustrated in the accompanying drawings. [Example]

[0177] The following detailed examples are intended to contribute to a better understanding of the present invention. However, the present invention is not limited to the examples. Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.

[0178] Example 1: Materials and Methods Construction of saRNAVRP (alphavirus replicon particle) The following section describes the construction of saRNAVRPs containing one or more heterologous nucleic acids expressing antigenic determinants. All other constructs described herein are made using similar methods.

[0179] Cloning of VEEV-based saRNA-launching replicon plasmids The recombinant alphavirus replicon particles VRP-BN001, -BN005, and -BN006 were all derived from the genome of the attenuated VEEV TC83 strain. To construct the replicon (or saRNA) in these particles, the open reading frame (ORF) downstream of the subgenomic promoter encoding the polyproteins C-E3-E2-6k-E1 in the VEEV TC83 genome was completely deleted, and a single A3G nucleotide substitution in the genomic 5' UTR was introduced to improve expression levels. The resulting replicon sequence (including the 5' UTR, ORFs encoding nonstructural proteins 1 to 4 (nsp1 to 4), the subgenomic promoter, and the VEEV TC83 genomic 3' UTR) was placed under the transcriptional control of the CMV promoter. A poly-A stretch and an HDV antigenomic ribozyme sequence were added downstream of the 3' UTR of the replicon to generate a polyadenylated replicon with a precise 3' end. Different antigens of interest were cloned and placed under the transcriptional control of a subgenomic promoter in this TC83-based replicon. VRPs containing VEEV TC83-based replicon constructs expressing different transgenes were generated.

[0180] 1) VRP-BN001 encodes a fusion protein (referred to herein as BBP-OVA) consisting of a tag blue fluorescent protein (BFP) ORF followed by a self-cleaving P2A peptide and an ovalbumin (OVA) ORF. The presence of the P2A peptide allows expression of OVA and BFP as independent proteins from a single mRNA.

[0181] 2) VRP-BN005 encodes an N-terminal FLAG-tagged (DYKDDDDK) version of the murine endogenous retroviral protein (MuLV) envelope glycoprotein 70 (gp70) containing two amino acid mutations (E552R and A558F) in the conserved immunosuppressive domain (ISD). Furthermore, the original signal sequence (aa 1-31) was replaced with an Ig-kappa (IgK) leader sequence for more efficient transport to the cell membrane.

[0182] 3) VRP-BN006 encodes a single-chain murine IL-12 protein in which the p40 and p35 subunits are linked by a 3x(GGGGS) linker sequence. Furthermore, the secretion signal of the p35 subunit (aa 1-22) has been deleted, resulting in the protein encoding only one secretion signal (from the p40 subunit).

[0183] The coding sequences of the above transgenes were codon-optimized and synthesized as DNA strings (including sequences necessary for cloning) and cloned into a replicon vector. Alternatively, sequences were PCR-amplified from existing constructs and cloned into the replicon.

[0184] Cloning of SFV-based saRNA-launching replicon plasmids The recombinant alphavirus replicon particle VRP-BN010 was derived from the Semliki Forest virus (SFV) genome. For the construction of this particle, the open reading frame (ORF) encoding the polyprotein C-E3-E2-6k-E1 downstream of the subgenomic promoter was deleted, except for the first 102 nucleotides (or aa 1-34) of the ORF encoding the capsid proteins. This sequence from the SFV capsid contains a known translational enhancer element sequence, which may increase transgene expression levels. To avoid fusion proteins between the transgene of interest and the N-terminus of the SFV capsid containing the enhancer sequence, a self-cleaving P2A peptide was introduced downstream of the truncated capsid. The resulting replicon sequence (including the 5' UTR, nsp1-4 ORF, subgenomic promoter, capsid translational enhancer element [C(enh)], P2A sequence, and 3' UTR of the SFV genome) was placed under the transcriptional control of the CMV promoter. A poly-A stretch and HDV antigenomic ribozyme sequence were added downstream of the 3' UTR of the replicon to generate a polyadenylated replicon with a precise 3' end. The BFP-OVA transgene was cloned and placed under the transcriptional control of the SFV subgenomic promoter, in-frame with the upstream capsid translation enhancer and P2A sequence, generating a polycistronic mRNA encoding C(enh)-P2A-BFP-P2A-OVA. The BFP-OVA ORF is identical to that encoded by VRP-BN001. The two P2A sequences in this mRNA have different nucleotide sequences to avoid recombination but encode the same protein sequence, allowing BFP and OVA to be expressed as independent proteins and separated from the C(enh) sequence.

[0185] Generation, purification, and titration of VRP-BN stock To generate the VRP-BN stock suspension, HEK293 T cells were transiently transfected with two packaging plasmids and a replicon plasmid encoding the transgene of interest.

[0186] In transfected cells, the CMV-driven replicon launches the first saRNA encoding nonstructural proteins (nsp1-4) and the antigen of interest, which is self-amplified within the transfected cells by the nsp1-4 alphavirus replication machinery. Two packaging plasmids, encoding capsid and envelope proteins separately, provide all the alphavirus structural proteins necessary for saRNA packaging and VRP formation. The VRP buds from the cells into the supernatant. The supernatant from the transfected cells is then harvested, concentrated, and sedimented by centrifugation through a 40% sucrose cushion. The resulting VRP pellet is resuspended in an appropriate buffer and titrated. The purified VRP stock is titrated in Vero cells by infection with serially diluted stocks. Transduced cells are detected by staining double-stranded RNA (dsRNA) replication intermediates using a dsRNA-specific mouse monoclonal antibody (J2, Jena Bioscience) and analyzed by flow cytometry.

[0187] The two packaging plasmids used to produce VEEV-VRP express: 1) A mutant VEEV TC83 capsid under the control of the CMV promoter. This mutant capsid (Cm) has an amino acid substitution that inactivates the nuclear localization signal (NLS) of the protein ( [ka] The array is [ka] mutated to 2) Under the control of the CMV promoter, the VEEV TC83 envelope protein (E3-E2-6k-E1 polyprotein) is processed by proteases within the cell to produce the mature VEEV TC83 envelope protein.

[0188] The two packaging plasmids used to produce SFV-VRPs encode a CMV-driven SINV / SFV “minigenome” consisting of the first 302 bp of the SINV genome [i.e., the 5′UTR and the known nsp1 replication enhancer element ( nsp1(enh)], the SFV 3′UTR followed by a poly-A stretch and an HDV antigenomic ribozyme sequence, and one of the following SFV structural proteins: 1) A mutant SFV capsid containing the S219A mutation, which abolishes the self-cleaving activity of the protein, was cloned in frame with a partial SINVnsp1(enh) sequence and separated by the F2A self-cleaving peptide [SINVnsp1(enh)-F2A-capsid]. The F2A peptide enabled expression of the SFV capsid separated from the SINVnsp1 sequence. 2) The SFV envelope protein (E3-E2-6k-E1 polyprotein) was cloned in frame with the partial SINV nsp1(enh) sequence and separated by the F2A self-cleaving peptide [SINVnsp1(enh)-F2A-E3-E2-6k-E1]. The F2A peptide enabled expression of the SFV envelope protein separated from the SINV nsp1 sequence.

[0189] Example 2: The level and duration of VRP-induced antigen expression varies among cell lines, but antigen expression is highest during the first 48 hours post-infection. To better understand VRP constructs and their potential as novel vaccination platforms, it is crucial to understand how these constructs function in various cell types. Therefore, we characterized two different VRPs (VRP-BN001 and VRP-BN010) in vitro using different cell lines. VRP-BN001 (VEEV-VRP-OVA-BFP) is derived from Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, while VRP-BN010 (SFV-VRP-OVA-BFP) is derived from Semiliqui Forest virus (SFV), an Old World alphavirus. Both of these constructs express blue fluorescent protein (BFP) and ovalbumin (OVA) as model antigens.

[0190] VRP-induced antigen expression in various cell lines may vary depending on the cell line type, as VRP tropism and type I interferon responses may differ between cell lines. Therefore, we investigated VEEV-VRP or SFV-VRP-induced OVA secretion in two different mouse cancer cell lines: 4T1 breast cancer cell line and CT26 colon cancer cells. In these experiments, cells were infected with 30 transducing units / cell (TU / cell) of VRP. After 1 hour, the VRP-containing cell medium was removed, and the cells were cultured for 5 days. The cell culture medium was replaced with fresh medium every 24 hours postinfection for 5 days and frozen at -20°C. At the end of the experiment, the amount of OVA protein in all cell supernatant samples was measured by ELISA to determine the kinetics of VRP-induced antigen expression after infection.

[0191] We observed that VEEV-VRP-OVA-BFP (VRP-BN001) and SFV-VRP-OVA-BFP (VRP-BN010) induced comparable amounts of OVA secretion by 4T1 cells, with SFV-VRP inducing slightly higher levels of OVA secretion than VEEV-VRP at 48 h. After this time point, OVA secretion began to decrease, and by 120 h, no OVA protein was detectable in the supernatant of infected cells (Figure 1A). Similarly, OVA secretion by VRP-infected CT26 cells was high for the first 2 days, but antigen expression began to decrease after 48 h. However, in this cell line, VEEV-VRP-induced OVA expression was stronger than SFV-VRP, and very low levels of OVA were still detectable in the supernatant of VEEV-VRP-infected cells at 120 h (Figure 1B). We also observed that VEEV-VRP-induced OVA secretion by CT26 cells was much higher than that of 4T1 cells (Figures 1A and 1B). This data indicates that antigen expression levels on VRPs can vary depending on the cell line. Furthermore, antigen expression is strongest during the first 48 hours post-infection and begins to decrease after this time point on both VRPs (Figure 1A and 1B).

[0192] Fibroblasts are an important component of the tumor microenvironment and may play an essential role in immunotherapy outcomes. For this reason, we also investigated antigen expression in the A31 mouse fibroblast cell line. Similar experimental procedures were performed, but these cells were infected with VRP at 10 and 100 TU / cell in addition to 30 TU / cell to confirm whether there was a correlation between antigen expression levels and the titer of VRP used for infection. We observed that higher titers of VEEV-VRP and SFV-VRP were associated with proportionally higher levels of OVA secretion upon infection. Consistent with our results from cancer cell lines, VRP-induced antigen expression was highest in fibroblasts during the first 48 h and began to decrease dramatically after this time point. At 120 h, no OVA protein was detectable in the cell supernatant. Finally, although the kinetics of antigen expression was comparable between cancer cells and fibroblasts, the amount of OVA secreted by fibroblasts was much lower than that of 4T1 and CT26 cell lines (Figure 1C).

[0193] In conclusion, our results from three different mouse cell lines indicate that antigen expression was highest during the first 48 hours after infection with VRP-001 and VRP-010 and began to decrease dramatically after this time point. At 120 hours, OVA protein in the cell supernatant was extremely low or undetectable. Furthermore, although the same VRP and titer were used for infection, differences in the amount of OVA secreted were detected among the tested cell lines. Potential reasons for this include differences in the proportion of VRP-infected cells, the cytotoxic effect of VRP, or the cessation of VRP-induced protein expression when cells induce a type I IFN response after infection.

[0194] Example 3: VRP infection leads to a decrease in cell viability of CT26, 4T1, and A31 cells in vitro. During our in vitro experiments using mouse 4T1 and CT26 cell lines, we observed reduced cell confluency and abnormal cell morphology after infection with VRP-BN001 and VRP-BN010. Based on these observations, we decided to investigate the effects of VRP on cell viability and proliferation using the CellTiter-Glo® 2.0 assay. This assay quantifies the amount of ATP as an indicator of metabolically active cells and can measure the number of viable cells in culture.

[0195] To this end, 4T1, CT26, and A31 cells were treated with TNE buffer as a control or infected with VEEV-VRP-OVA-BFP (VRP-BN001) and SFV-VRP-OVA-BFP (VRP-BN010) at 0-80 TU / cell. CellTiter-Glo® 2.0 assays were performed 24 or 48 hours postinfection.

[0196] The results showed that TNE-treated samples already had reduced viability compared to cells cultured in cell culture medium containing 10% FCS, suggesting that the TNE buffer itself inhibits cellular metabolism, resulting in reduced cell confluency compared to untreated cells. Consistent with previous observations, we confirmed that 4T1 cells infected with higher concentrations of VRP-BN001 and VRP-BN010 (80 TU / cell) showed reduced cell viability. This effect was more pronounced in CT26 cells, especially after 48 hours.

[0197] Interestingly, we were able to detect more viable 4T1 cells at 48 h. Cells maintained in cell culture medium with FCS also exhibited higher luminescence at this time point, leading us to conclude that this was due to cell proliferation. In contrast, this difference was not detected in the luminescence values ​​of CT26 cells. This observation is expected, given that 4T1 cells are more aggressive and proliferate faster in culture than CT26 cells. Furthermore, the cytotoxicity of VRP was weaker in A31 mouse fibroblasts at both time points compared to the other two cell lines we used, which is consistent with our previous observations.

[0198] In conclusion, we confirm previous findings showing that in the cell lines used, VRP infection causes a decrease in cell viability and that the cytotoxic effect of VRP varies depending on the cell type.

[0199] Example 4: Detection of VEEV-VRP biodistribution by qPCR 6 hours after intratumoral administration Our VRP platform is a promising vaccine candidate based on the efficient activation of antigen-specific T cells in both tumor and infection models, as well as the strong antibody responses observed in EBV-NHP studies. However, our knowledge of the mechanism by which VRP induces immune responses is limited. Due to its smaller size and possibly different cell tropism, we hypothesize that the biodistribution of VRP and its mechanism of action for activating T and B cells may differ from that of our vaccine platform, MVA. Understanding how VRP elicits immune responses, which cell types it infects, and where VRP localizes after local administration may be useful in enhancing VRP-induced antigen-specific adaptive immune responses when used as an allogeneic immunization or a heterologous immunization in combination with other vaccine platforms, such as MVA.

[0200] To address this issue, we investigated whether VRP reaches distant organs after intratumoral (IT) administration. Mice bearing 4T1 tumors were administered TNE buffer as a control or 1 × 10 ovalbumin (OVA)-expressing VRP. 8 Transfection units (TU) of VEEV-VRP-OVA-BFP (VRP-BN001) were administered to the animals. Animals were sacrificed 6 or 24 hours after IT administration, and organs were harvested. mRNA was extracted and qPCR analysis was performed to detect the expression of OVA mRNA encoded by VRP.

[0201] As expected, OVA expression was not detected in TNE-treated control samples, but in VRP-treated mice, the highest expression was observed in tumor and tumor-draining lymph node (TdLN) samples 6 hours after IT administration. At this time point, OVA expression was also observed in the spleen of one mouse, whereas no signal was detected in non-draining lymph node (non-dLN), lung, or liver samples (Figure 3A). Interestingly, OVA expression was still detectable in tumor samples at 24 hours, albeit at lower levels compared to earlier time points (Figure 3B).

[0202] Our results indicate that VRP can be detected not only in tumors but also in distant organs after local injection. The highest OVA mRNA levels were detected 6 hours after IT injection, and the signal significantly decreased at 24 hours. The strong signal observed in TdLNs may be due to the small particle size of VRP. Furthermore, VRP may be more efficient at infecting target cells and expressing antigens.

[0203] Example 5: Detection of VEEV-VRP biodistribution by immunofluorescence (IF) staining 6 hours after intratumoral administration As a next step, we decided to employ a second method to confirm the biodistribution of VRP. To this end, B16.F10 tumor-bearing mice were administered TNE buffer as a control or 1 × 10 VRP expressing blue fluorescent protein (BFP) as an antigen. 8 VEEV-VRP-OVA-BFP (VRP-BN001) was administered to the tumors. Six hours after IT administration, the animals were euthanized, and the tumors and tumor-draining lymph nodes (TdLNs) were harvested, pre-fixed in paraformaldehyde to preserve the BFP signal, and cryopreserved. Seven-µm-thick tissue sections were prepared and stained with FITC-conjugated CD45.2 antibody to detect leukocytes. Because the BFP signal itself was too weak to visualize, we used an anti-tagRFP antibody, which binds to the BFP protein encoded by VRP, and a secondary antibody labeled with AF647 (because the anti-tagRFP antibody was not fluorescently labeled). Nuclei were stained with DAPI.

[0204] Six hours after IT administration, BFP signals were observed in both the tumor and TdLN (Fig. 4A, B). In the tumor, both FITC-positive and -negative cells expressed BFP. + Therefore, VRP was associated with tumor cells and tumor-infiltrating CD45 +These findings suggest the possibility of infection of leukocytes and other components of the tumor microenvironment (TME), such as fibroblasts. An additional hypothesis is that some immune cells (antigen-presenting cells (APCs)) may have become positive due to phagocytosis of VRP-infected tumor cells (Figure 4A). In TdLNs, the BFP signal was stronger than in the tumor tissue and was mainly localized in the capsular and subcapsular regions of the lymph nodes (Figure 4B). As expected, no BFP signal was detected in TNE-injected mice (data not shown).

[0205] In summary, this data supports previous findings that detected VRP-induced OVA expression in both tumor and TdLN samples 6 h after local administration of VEEV-VRP-OVA-BFP.

[0206] Example 6: VEEV-VRP expressing tumor-associated antigen (TAA) Gp70 (VRP-BN005) induces a strong anti-tumor immune response in the CT26 colon cancer model. We previously demonstrated VRP-induced antigen-specific T cell responses in naive mice in immunogenicity experiments. Therefore, we decided to investigate whether intratumoral (IT) administration of VRP enhances antitumor immune responses in tumor-bearing mice. To this end, the Vaccine Development Department generated VEEV-VRP expressing the endogenous retroviral element Gp70 (VEEV-VRP-Gp70, VRP-BN005, hereafter abbreviated as VRP-Gp70) as a tumor-associated antigen (TAA). To examine its antitumor efficacy, we used the CT26 mouse colon cancer model. Treatment of mice bearing subcutaneously implanted CT26.WT tumors began when the tumors reached approximately 5.5 × 5.5 mm in size. Mice were administered 1.00E+08 TU of VRP-Gp70 via intratumoral (IT) injection. This time point was defined as day 0, and booster inoculations were administered on days 4 and 7, as shown in Table 1.

[0207] [Table 2]

[0208] IT treatment with PBS slightly delayed tumor growth, reflecting the immunogenicity of CT26.WT tumors. Surprisingly, mice injected three times with VRP-Gp70 eradicated five of eight tumors (Figure 5A). Analysis of CD8 T cell responses in peripheral blood 2 days after the last injection revealed similar CD8 T cell frequencies in PBS- and VRP-Gp70-treated mice (Figure 5B). However, increased IFN-γ production by CD8 T cells was detected in the VRP-Gp70-treated group upon restimulation with the Gp70-specific peptide AH-1, and a similar trend was observed in the two PBS-treated animals (Figure 5C). In some, but not all, cases, this CD8 T cell response correlated with antitumor efficacy, as shown by the dashed tumor growth graph.

[0209] Our experiments highlight tumor antigen-encoding VRPs as a novel platform for inducing therapeutic responses against tumors and show that IT administration of VRP-Gp70 dramatically enhances tumor rejection and systemic CD8 T cell responses against endogenous antigens of CT26.WT tumors.

[0210] Example 7: VRP-BN005 (VRP-Gp70) induces anti-tumorigenic immune responses in the B16.F10 melanoma tumor model. Next, we investigated the therapeutic potential of VRP-Gp70 in a non-immunogenic murine B16.F10 melanoma tumor model. Treatment of mice bearing subcutaneously implanted B16.F10 tumors began when tumors reached approximately 4.5 × 4.5 mm in size. This time point was defined as day 0, and booster inoculations were administered on days 5 and 8, as shown in Table 2.

[0211] [Table 3]

[0212] IT treatment with PBS did not result in any tumor growth control, indicating that this tumor was differentially immunogenic compared to CT26.WT, which was previously used to confirm the efficacy of this construct. Interestingly, three IT doses of VRP-Gp70 induced tumor growth suppression, halting tumor growth in five of eight mice in this group (Figure 6A). Furthermore, analysis of peripheral blood CD8 T cell responses 4 days after the last injection revealed that both CD8 T cell frequency and IFN-γ production by CD8 T cells increased in the VRP-Gp70-treated group upon restimulation with the Gp70-specific peptide p15E (Figure 6B, C).

[0213] Overall, our results demonstrate that repeated local administration of VRP-Gp70 enhances tumor growth suppression not only against immunogenic CT26 tumors but also against non-immunogenic, refractory B16.F10 melanoma, suggesting that our VRP platform is an excellent vaccine candidate in immuno-oncology.

[0214] Example 8: Local administration of VRP-BN005 (VRP-Gp70) provides long-term protection in mice against tumor re-challenge. We have previously demonstrated the therapeutic efficacy of intratumoral administration of VRP expressing the endogenous retroviral element Gp70 (VRP-Gp70; VRP-BN005) in two different syngeneic tumor models: the CT26 colon cancer model and the B16.F10 melanoma. The antitumorigenic responses induced by these treatments varied between the two tumor models due to their diverse immunogenicity, but the most promising results were observed in the CT26.WT model. In contrast to the B16.F10 tumor, the CT26 colon cancer model is classified as a "hot tumor," exhibiting abundant lymphocyte infiltration and shown to be responsive to checkpoint inhibitor treatment (Mosely et al., Cancer Imm Res. 2016).

[0215] As previously reported, repeated IT injections of VRP-Gp70 resulted in the eradication of CT26.WT tumors in 5 of 8 mice, and in some animals, VRP-Gp70-induced antigen-specific CD8 T cell responses correlated with the antitumor effect, as shown by the tumor growth graph with dashed lines (Figure 5A). We next asked whether this strong antitumor response provided long-term protection to the mice.

[0216] To answer this question, previously cured animals were used in a rechallenge experiment and administered the same cancer cells (CT26) subcutaneously on the contralateral side. Tumor growth was monitored for 33 days after tumor injection. As shown in Figure 7A, tumors developed only in untreated mice, whereas no tumor growth was observed in cured mice. This data demonstrates that local VRP-Gp70 treatment induced a memory response and long-term protective effects in cured mice.

[0217] Next, we investigated the CD8 T cell response to Gp70-specific AH-1 peptide stimulation. To this end, blood was analyzed 6 days before and after tumor rechallenge, and splenocytes were analyzed on day 33. In cured mice that rejected tumors, there was an increase in the percentage of total CD8 T cells and AH1-specific CD44 T cells 6 days before and after rechallenge. + IFNγ + An increase in CD8 T cells was observed (Fig. 7B, C), suggesting activation of antigen-specific memory CD8 T cells in cured mice upon tumor rechallenge.

[0218] Because tumor growth was not observed in the rechallenge group, we investigated the contribution of memory T cell subsets to this process. We analyzed peptide-specific CD8 T cell responses and memory T cell subsets in spleen, tumor-draining lymph node (TdLN), non-draining lymph node (Non-dLN), and skin samples collected from mice 33 days after tumor rechallenge. For these analyses, skin tissue was collected from the contralateral injection site, and in cured mice, Non-dLN was removed from the area close to the previous tumor injection site. AH-1 pentamer staining was performed to detect CD8 T cells specific for the AH-1 peptide. In skin samples from VRP-Gp70-cured mice, CD44 + AH-1 + The percentage of CD8 T cells was the highest (Figure 8A). Increases in these cells were also detected in other organs of cured mice compared to control mice (Figure 8A). To identify the memory precursor CD8 T cell subset (MPEC), CD127 and KLRG1 were used. + CD44 + AH-1 + CD127 + KLRG1 - These cells were defined as the CD8 MPEC population. This population was further analyzed to identify central memory T cells (T CM )(CD127 + CD62L + ) and effector memory T cells (T EM )(CD127 + CD62L - ) was identified. As is known, T CM cells reside in lymphoid tissues and are reactivated upon secondary infection or challenge, whereas T EM CD8 T cells circulate in various tissues and have cytotoxic properties. CM We found that the number of CD8 T cells increased in the spleen and TdLN of cured mice (Figure 8B). Furthermore, in cured mice, the number of CD8 T cells in the TdLN, spleen, and skin was significantly increased. EM Importantly, there was a similar increase in the proportion of T EM The proportion of these organs was significantly higher than that of other organs examined (Fig. 8C).

[0219] Tissue-resident memory T cells (T RM ) have recently been identified. These cells reside in tissues (skin, lung, etc.) and do not recirculate. RM The cells are functionally and transcriptionally T CM Cells and T EM Unlike cells, mainly T EM However, they are not associated with T CM It may also originate from cells (Enamorado et al., Nat Commm. 2017). RM The primary role of these cells is to protect epithelial tissues from inflammation or infection. The primary cell surface markers used to define these cells are CD103, CD49a, and CD69, and these markers can vary depending on the tissue. CD103 is the most common marker associated with most CD8 + T RM It is expressed by T cells and has been suggested to be involved in the homing of T cells to epithelia. The C-type lectin CD69 plays a role in the retention of cells in tissues. Furthermore, CD49a is expressed by two T RM It is used to distinguish cell subsets. It has been shown that CD49a-expressing cells produce perforin and IFNγ (Mami-Choaib et al., J Immunother Cancer. 2018). The present invention is not bound by or limited to a particular scientific principle or mechanism of action, and since no tumor growth was observed after tumor rechallenge in mice cured with VRP-Gp70, it is possible that T cells may play a role in preventing the growth and / or eliminating tumor cells at the injection site. RM For this purpose, we investigated the presence of CD8 T cells. EM The expression of CD69 and CD103 was examined in the subsets. As shown in Figures 8D and 8E, CD69 and CD103 were detected in the skin samples from healed mice. + CD103 + and CD69 + CD103 - T RM In the skin, both CD69 + CD103- T RM The percentage of cells that are CD69 + CD103 + was observed to be much higher than that of cells, suggesting that these two populations may be functionally distinct (Fig. 8D, E).

[0220] We further analyzed CD4 T cells in TdLN, non-dLN, spleen, and skin samples 33 days after tumor rechallenge. Because we do not have the tools to detect antigen-specific CD4 T cells by flow cytometry, we were unable to detect the proportion of these cells. However, CD44 + Once identified, CD4 T cells were identified using the same gating strategy as for CD8 T cells as described above. CM and T EM The subset was further identified. CD4 T cells were more abundant in the TdLN tissues of healed mice compared with control mice. CM Although we detected increased accumulation of CD4 T subsets, we were unable to detect these cells in the skin (Fig. 9A). EM The elevated accumulation of CD69 subsets was more pronounced in tissues of healed mice compared to controls, with the highest accumulation detected in the skin (Figure 9B). Furthermore, the elevated accumulation of CD69 subsets in all organs of healed mice was significantly higher than that in controls. + CD103 + and CD69 + CD103 - T RM The frequency of these two distinct T cells was similar. RM The infiltration rates of the subsets were similar in the skin of healed mice (Fig. 9C, D).

[0221] In summary, our results demonstrate that repeated IT administration of VRP-Gp70 not only resulted in tumor elimination in CT26.WT tumor-bearing mice, but also provided long-term protection against rechallenge with the same tumor cell line. The peptide-specific CD8 T cell responses upon AH-1 peptide restimulation in cured mice increased after tumor rechallenge, peaking on day 33. This suggests that subcutaneous injection of CT26.WT cells induced antigen-specific CD8 T cell responses in cured mice. The presence of peptide-specific CD8 T cells in different organs was identified by AH-1 pentamer staining. The highest amount of AH-1 + Specific CD8 T cells were detected in the skin of healed mice, and they were primarily T EM Further investigation of this population revealed that it was composed of CD69 + CD103 - T RM It was observed that CD69 cells accounted for the largest proportion of this population. + CD103 + T RM It can also detect T cells. EM and T RM These results suggest that T cells play an important role in the elimination and / or growth inhibition of subcutaneously injected tumor cells. RM The group circulates T EM It is not clear whether they arose from a pool or migrated from the site of previous tumor growth.

[0222] For CD4 T cells, no tetramer or pentamer works to confirm antigen-specific CD4 T cell responses as does the CD8 counterpart. Furthermore, we found that CD4 T cells were expressed in the organs of all cured mice. EM We observed an increase in CD4 T cells, which was most pronounced in the skin, similar to that observed for CD8 T cells. Indeed, half of the skin-infiltrating CD4 T cells were T RM These cells expressed related markers, such as CD103 and / or CD69. +T RM As CD4 + T RM These results suggest that IL-1 plays a role in controlling tumor regrowth upon rechallenge.

[0223] Example 9: IT administration of VRP-BN005 (VRP-Gp70) modulates the tumor microenvironment (TME), thereby enhancing infiltration of T lymphocytes and NK cells. One strategy for cancer immunotherapy is to induce reprogramming of the immunosuppressive tumor microenvironment (TME) to a proinflammatory environment, allowing the development of innate and adaptive antitumor immune responses. As previously reported, intratumoral injection of rMVA induces inflammation in the TME, and adjuvant administration of MVA-OVA with the costimulatory receptor 4-1BBL increases inflammatory cytokine concentrations in tumors, thereby significantly enhancing therapeutic efficacy (Hinterberger et al., JITC 2021). Furthermore, various studies have shown that replication-competent oncolytic viruses induce inflammation in the TME (Boardois-Darygneault, Science Trans. Med 2018). Based on this information and the results demonstrating the efficacy of intratumoral administration of VRP-Gp70 in inducing antitumor immune responses in two different tumor models, we decided to investigate how local administration of VRP affects immune cell infiltration in the tumor microenvironment (TME). Therefore, we performed a detailed analysis of the infiltration of innate and adaptive immune responses within tumors at different time points (days 1 and 7) after IT injection into B16F10 melanoma tumors. First, we found that a single IT injection of VRP-Gp70 could result in significantly smaller B16.F10 tumors one week later (Figure 10A). In addition, mice receiving an IT injection of VRP-Gp70 had significantly lower CD45 expression levels on day 1 compared to controls. + Leukocyte infiltration into the tumor was high, and as expected, this increase was more pronounced on day 7 (Figure 10B). + and CD8 + Both CD8 T cells and CD8 T cells were increased in the TME 1 week after immunization, as indicated by cell counts. + T cells were CD45 in the tumor by day 7+ Furthermore, the number of NK cells in the TME was higher in VRP-Gp70-treated mice than in the saline-treated group on days 1 and 7 (Figure 10B).

[0224] In summary, our data show that a single IT immunization of VRP-Gp70 significantly reduced B16.F10 tumor weights 1 week after treatment. Furthermore, CD45 upregulation was observed on day 1 in VRP-Gp70-immunized mice. + Leukocyte infiltration was induced to a greater extent, and cellular quantification revealed that NK cells expressed CD45 in the tumor microenvironment (TME) of VRP-Gp70-immunized mice at this time point. + Furthermore, VRP-Gp70-treated mice showed a significant increase in CD4+ cells by day 7. + and CD8 + Enhanced T cell infiltration indicates that a single IT injection of our VRP construct induces the infiltration of cells responsible for cancer cell elimination, which may explain why tumors are significantly smaller after 1 week.

[0225] Example 10: Encoding IL-12 enhances VRP-induced immune cell activation in vitro. IL-12 inhibits CD8 + Cytotoxic T cells, type 1CD4 + It is an important cytokine with potent effects on helper T cells and NK cells. IL-12 mRNA or IL-12-modified tumor-specific CD8 +Intratumoral (it) administration of T cells has been shown to be effective in multiple preclinical models (Etxeberria et al., Cancer Cell 2019; Hewitt et al., Trans Cancer Mec and Ther. 2020). Furthermore, it injection of plasmid DNA encoding IL-12 resulted in significant clinical responses in patients with metastatic melanoma, and the treatment was well tolerated (Heinzerling et al., Hum Gene Ther. 2005). Based on its important role in regulating the immune system and its efficacy in inducing potent antitumor responses against various cancer types, we decided to generate a VEEV-VRP expression vector expressing a single-chain IL-12p40p35 fusion protein (IL-12sc) and evaluate its efficacy in vitro and in vivo. As a first step, we evaluated the biological activity of IL-12-expressing VEEV-VRP (VEEV-VRP-IL12; VRP-BN006; hereafter referred to as VRP-IL12) constructs by infecting mouse splenocytes in vitro. As controls, recombinant IL-12p70 (rIL-12p70) was used to determine IL-12-specific effects, and VEEV-VRP (VRP-BN015; hereafter referred to as VRP) was used to evaluate the effects of VRP without adjuvant. Uninfected splenocytes were used as a negative control. Eighteen hours after infection, splenocytes were analyzed by measuring the surface expression of CD69 or by intracellular staining for granzyme B and IFNγ, respectively. Our results showed that VRP-IL12 infection significantly increased CD8 expression in vitro compared with VRP infection. +This resulted in increased activation and cytotoxicity of both T cells and NK cells (Figures 11A and 11B). Furthermore, analysis of cell supernatants 18 hours later showed enhanced secretion of IL-12 and IL-12-induced IFNγ in VRP-IL12-infected cells compared with VRP-infected cells (Figures 11C and 11D). Furthermore, VRP-IL12 infection also increased the secretion of other cytokines and factors, including IL-6, IL-10, GM-CSF, and TNFα (Figures 11E–H). Overall, our findings validated the biological activity of VRP-IL12 and demonstrated that encoding IL-12 enhanced the in vitro immune cell induction capacity of the VRP vector.

[0226] Example 11: IT administration of VRP-IL12 improves tumor growth control and survival in the poorly immunogenic B16.F10 tumor model without causing toxicity. Next, we evaluated the toxicity and therapeutic efficacy of VRP-IL12 (VRP-BN006) in vivo using B16.F10 tumor-bearing mice. Based on previous results demonstrating the promising antitumor efficacy of VRP-gp70 (VRP-BN005) in this tumor model, we hypothesized that the therapeutic efficacy of VRP might be enhanced by adding IL-12 as an adjuvant to VRP. To this end, treatment of mice bearing subcutaneously implanted B16.F10 tumors was initiated when tumors reached approximately 5 × 5 mm in size by intratumoral (IT) injection of increasing titers of 1.00E+08TU VRP or VRP-IL12. This time point was defined as day 0, and booster inoculations were administered on days 5 and 8, as shown in Table 3.

[0227] [Table 4]

[0228] Control mice treated with TNE buffer had to be sacrificed early in the experiment due to poor condition, but 1 × 10 8IT administration of TU VRP already showed mild tumor growth inhibition in 4 of 5 mice (Figures 12A and 12B). VRP titers were 1 × 10 6 , 1×10 7 , or 1 × 10 8 VRP-IL12 was administered intratumorally. Interestingly, no significant differences in the antitumor effects of VRP-IL12 were observed between different doses, and the lowest dose was already strong enough to promote antitumor responses in mice (Figures 12C-12F). Furthermore, it was clearly demonstrated that the addition of IL-12 significantly improved the antitumor effect of VRP and increased mouse survival compared with VRP-treated control mice. Very importantly, no IL-12-related toxic effects were observed, even after administration of the highest dose of VRP-IL12.

[0229] In summary, our results demonstrate that IT immunization with high-titer VRP expressing IL-12 does not result in toxic effects in B16.F10 tumor-bearing mice. This property of VRP-IL12 is crucial for vaccine platforms, as several clinical trials have already reported that high IL-12 expression can lead to inflammatory syndromes. Furthermore, the lower titer (1 × 10) of VRP-IL12 resulted in no toxic effects in B16.F10 tumor-bearing mice. 6 Even IT administration of VRP-IL12 (TU) can promote a strong antitumor response, which is not significantly improved by administration of higher titers of VRP. This suggests that lower VRP titers can be used for effective treatment, which may also be more beneficial for stimulating immune responses primarily against the antigen of interest rather than against the VRP itself.

[0230] Example 12: Combination of VRP-IL12 with VRP-expressing TAA induces a stronger anti-tumor response in the low-immunogenic B16.F10 tumor model. Because VRP-IL12 did not express tumor-associated antigens (TAA), combining VRP expressing TAA Gp70 and IL-12 resulted in tumor-specific CD8+ cells in the B16.F10 melanoma model. +We hypothesized that antitumor immune responses may be enhanced through the induction of T cell responses. To this end, B16.F10 tumor-bearing mice received three consecutive IT doses of TNE buffer, a combination of VRP-BN015 (VRP) and VRP-BN005 (VRP-Gp70), or a combination of VRP-BN005 (VRP-Gp70) and VRP-BN006 (VRP-IL12). In all VRP-treated groups, the total VRP titer used was 1 x 10 per mouse. 8 Immunization with VRP-BN015 + VRP-BN005 induced mild tumor growth control compared to the TNE buffer control group, whereas the combination of VRP-005 and VRP-006 showed more potent tumor growth control, with complete tumor regression observed in 1 out of 5 mice (Figure 13A-D).

[0231] These results demonstrated that IT coadministration of VRP-IL12 and VRP-Gp70 was highly effective in the treatment of low-immunogenic B16.F10 tumors, resulting in tumor regression in some treated mice. This finding suggests that the presence or absence of the tumor antigen Gp70 may not be critical for VRP-IL12-induced antitumor efficacy in this model, indicating that induction of tumor-specific T cells may not be the primary mechanism of action. Rather, IL-12 may act primarily through the innate branch of the immune system, for example, by enhancing NK cell function.

[0232] Example 13: Comparison of different IT immunization schedules against VRP-IL12. After demonstrating the enhanced antitumor effect of VRP when adjuvanted with IL-12, we investigated whether the outcome of this therapy might differ by using a different immunization schedule. To test this, B16.F10 tumor-bearing mice were immunized with 1 × 10 8Mice were vaccinated intratumorally (IT) with TU's VRP (VRP-BN0015) or VRP-IL12 (VRP-BN006), and the day of the first immunization was accepted as day 0. Booster inoculations were repeated either on days 5 and 8, or on days 7 and 14, as shown in Table 4. As a negative control, mice were injected IT with TNE buffer on days 0, 5, and 8.

[0233] [Table 5]

[0234] Consistent with our previous findings, TNE buffer-treated control mice were sacrificed early in the experiment due to rapid tumor progression (Fig. 14A). However, IT administration of VRP on days 0, 5, and 8 induced mild tumor growth inhibition in 4 of 5 mice (Fig. 14B). Furthermore, IT administration of VRP-IL12 on days 0, 5, and 8 improved tumor growth inhibition and survival compared with VRP-treated mice (Fig. 14C). When IT immunization with VRP and VRP-IL12 was performed on a weekly schedule, both administrations induced tumor growth control similar to that of mice immunized on days 0, 5, and 8 (Fig. 14D, E). However, weekly administration resulted in shorter median survival. In the VRP-treated group, median survival was 29 days vs. 21 days on days 0, 5, and 8, and 0, 7, and 14, respectively, after immunization. In the VRP-IL12 group, median survival was 56 days vs. 46 days. TNE-treated mice had a median survival of 21 days, indicating that weekly VRP administration abolished the survival advantage of VRP treatment (Figure 14F).

[0235] In summary, we confirmed that IL-12-encoding VRP enhanced tumor growth inhibition and prolonged survival in mice treated with VRP, even when administered once a week. On the other hand, repeated IT treatment with VRP or VRP-IL12 at shorter intervals may be more effective than weekly administration in prolonging survival. However, this experiment should be repeated with larger groups to reach a definitive conclusion.

[0236] array SEQ ID NO: 1 BRP-OVA nucleotide sequence

[0237] SEQ ID NO: 2 BFP-OVA amino acid sequence MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM ALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLNGSGATNFSLLKQAGDVEENPGPASMGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLP GFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMAS EKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP

[0238] SEQ ID NO: 3 Mouse GP70 nucleotide sequence

[0239] SEQ ID NO: 4 Mouse GP70 amino acid sequence METDTLLLWVLLLWVPGSTGDYKDDDDKVALGNSPHQVFNLSWEVTNGDRETVWAITGNHPLWTWWPDLTPDLCMLALHGPSYWGLEYRAPFSPPPGPPCCSGSSDSTSGCSRDCEEPLTSYTPRCNTAWNRLKLSKVTHAHNEGFYVCPGPHRPRWARSCGGPES FYCASWGCETTGRASWKPSSSWDYITVSNNLTSDQATPVCKGNKWCNSLTIRFTSFGKQATSWVTGHWWGLRLYVSGHDPGLIFGIRLKITDSGPRVPIGPNPVLSDRRPPSRPRPTRSPPPSNSTPTETPLTLPEPPPAGVENRLLNLVKGAYQALNLTSPDKTQE CWLCLVSGPPYYEGVAVLGTYSNHTSAPANCSVASQHKLTLSEVTGQGLCIGAVPKTHQVLCNTTQKTSDGSYYLAAPTGTTWACSTGLTPCISTTILNLTTDYCVLVELWPRVTYHSPSYVYHQFERRAKYKREPVSLTLALLLGGLTMGGIAAGVGTGTTALVA TQQFQQLQAAMHDDLKEVEKSITNLEKSLTSLSEVVLQNRRGLDLLFLKRGGLCAFLKEECCLYADHTGLVRDSMAKLRERLSQRQKLFESQQGWFEGLFNKSPWFTTLISTIMGPLIILLLILLFGPCILNRLVQFIKDRISVVQALVLTQQYHQLKTIGDCKSRE

[0240] SEQ ID NO: 5 Mouse IL-12 nucleotide sequence

[0241] SEQ ID NO: 6 Mouse IL-12 amino acid sequence MCPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSG RFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFF VRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLK TCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

Claims

1. 1. A self-amplifying RNA (saRNA) for use in treating a tumor, the saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA increases the inflammatory response in the tumor, reduces the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or injection of saRNA that does not contain a nucleic acid encoding IL-12, and wherein the saRNA is administered intratumorally.

2. 2. The saRNA for use according to claim 1, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 2 (TRP2), brachyury, melanoma preferentially expressed antigen (PRAME), folate receptor 1 (FOLR1), human endogenous retrovirus-K envelope (HERV-K-env), human endogenous retrovirus-K-gag (HERV-K-gag), and combinations thereof.

3. A method for inhibiting tumor growth and / or increasing survival in a subject having a tumor, the method comprising intratumorally administering to the subject a saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA), a second nucleic acid encoding IL-12, and optionally a second nucleic acid encoding CD40L, wherein the intratumor administration of the saRNA increases the inflammatory response in the tumor, reduces tumor growth and / or size, and / or increases overall survival of the subject compared to injection of a saRNA that does not include a nucleic acid encoding IL-12 or saRNA alone.

4. 4. The method of claim 3, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), brachyury, PRAME, FOLR1, HERV-K-env, HERV-K-gag, and combinations thereof.

5. The method according to any one of claims 3 to 4, wherein the subject is a human.

6. 1. A method for reducing tumor size or growth and / or increasing survival in a tumor-bearing subject, the method comprising intraperitoneally administering to the subject saRNA comprising a first nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the administration of the saRNA increases natural killer (NK) cell responses and enhances CD8 T cell responses specific to the TAA compared to expected results from injection of saRNA alone.

7. 7. The method of claim 6, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), brachyury, PRAME, FOLR1, HERV-K-env, HERV-K-gag, and combinations thereof.

8. The method of any one of claims 6 to 7, wherein the subject is a human and the tumor is in the abdominal cavity.

9. The method of any one of claims 6 to 8, further comprising intratumorally administering to the subject an saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA), wherein the TAA may be the same TAA as the TAA of claim 7 or may be a different TAA.

10. A method for causing an increased inflammatory response in an intraperitoneal tumor in a subject, the method comprising intraperitoneally administering to the subject an saRNA comprising a first nucleic acid encoding a heterologous tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the intraperitoneal administration of the saRNA results in an increased inflammatory response in the tumor compared to the inflammatory response that would be induced by non-intraperitoneal administration of the saRNA virus alone.

11. 11. The method of claim 10, further comprising administering to the subject a boosting dose of the same saRNA intraperitoneally.

12. 1. A saRNA comprising (a) a first nucleic acid encoding a tumor-associated antigen (TAA) and (b) a second nucleic acid encoding IL-12, wherein intratumoral administration of the saRNA increases the inflammatory response in a tumor, decreases the growth rate and / or size of the tumor, and / or increases overall survival of the subject, compared to non-intratumoral administration of the saRNA or administration of a saRNA that does not contain a nucleic acid encoding IL-12.

13. 13. The saRNA of claim 12, wherein the TAA is selected from the group consisting of carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), human epidermal growth factor receptor 2 (HER-2), survivin, tyrosine-linked protein 1 (TRP1), tyrosine-linked protein 1 (TRP2), brachyury, FOLR1, PRAME, HERV-K-env, HERV-K-gag, p15, and combinations thereof.

14. A pharmaceutical combination comprising the saRNA of claim 12 and a pharmaceutically acceptable carrier.

15. A pharmaceutical combination comprising the saRNA of claim 12 and a second saRNA comprising a nucleic acid encoding a tumor-associated antigen (TAA) and a second nucleic acid encoding IL-12, wherein the TAA may be the same TAA as that of claim 13 or may be a different TAA.

16. 16. The pharmaceutical combination of claim 15, wherein the second saRNA comprises a first nucleic acid encoding a TAA that is a different TAA than the TAA encoded by the saRNA of claim 15.

17. A method for stimulating an immune response in a subject having multiple tumors, comprising administering locally (intratumorally) to some, but not all, of the tumors in the subject an saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12, wherein an immune response to the TAA is stimulated in the subject.

18. A method of treating a subject having at least one inaccessible tumor and at least one accessible tumor, comprising administering locally (intratumorally) to at least one accessible tumor of the subject an saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12, thereby reducing or stopping the growth of the inaccessible tumor.

19. A method for preventing or reducing the extent of tumor recurrence or metastasis in a subject having at least one tumor, the method comprising administering intratumorally or intraperitoneally to at least one tumor in the subject an saRNA comprising at least one first nucleic acid encoding a TAA and a second nucleic acid encoding IL-12, thereby reducing or stopping the growth of inaccessible tumors.

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