Cytokine immunotherapy

Abstract

To provide improved cytokine immunotherapy for cancer and / or inflammatory diseases. [Solution] This disclosure provides a novel immunologically active alphavirus repliconvector containing nucleic acids encoding alphavirus non-structural proteins nsp1-4 and cytokine proteins / polypeptides. The alphavirus repliconvector of this disclosure is useful for the treatment of cancer and / or inflammatory diseases.

Description

[Technical Field]

[0001] This disclosure relates, in general, to the field of cytokine immunotherapy for cancer and / or inflammatory diseases, and more particularly to alphavirus replicaconvectors containing cytokines useful for the treatment of cancer and / or inflammatory diseases. [Background technology]

[0002] Cytokines are molecular messengers of innate and adaptive immunity, enabling immune system cells to exchange information at close range and playing an indispensable role in regulating all aspects of the immune response, including lymphoid development, homeostasis, differentiation, tolerance, and memory. Given that the immune system can recognize and destroy cancer cells, there has been considerable interest in using cytokines for cancer treatment for decades.

[0003] Preclinical studies using interferon-alpha (IFNa), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-2, IL-12, IL-15, and IL-21 have demonstrated efficacy in multiple mouse cancer models.

[0004] IFNa was the first cytokine approved in 1986 for the treatment of human cancer, pilocytic cell leukemia (HCL). After evaluation of numerous treatment regimens, high-dose IL-2 (HDIL-2) was approved in 1992 for the treatment of metastatic renal cell carcinoma (mRCC) and in 1998 for the treatment of metastatic melanoma (MM). Since its initial approval, IFNa has been indicated for follicular lymphoma, as an adjuvant for melanoma, in combination with bevacizumab for mRCC, and for AIDS-associated Kaposi's sarcoma. Nevertheless, cytokines as monotherapy have not lived up to initial expectations.

[0005] Strategies to address local effects, in contrast to systemic effects, have included local or lumen administration of cytokines to enhance the mild outcomes of cytokine monotherapy, and transduction of cytokine-encoding genes into stimulated or effector cells via plasmid or viral delivery. Other novel approaches include structure-based cytokine manipulation to generate "superkines" with increased binding affinity to selective receptors to enhance antitumor responses and proportionally reduce Treg stimulation. The development of chimeric antibody-cytokine fusion proteins and the injection of cytokines with associated anti-cytokines have improved their tumor localization and pharmacokinetics compared to as-native molecules. Further clinical studies have shown that cytokines, when combined with anti-cancer vaccines, checkpoint inhibitor (CPI) antibodies (anti-CTLA-4 or anti-PD-1 / PD-L1), and cytokine injections when combined with cancer-targeting monoclonal antibodies, enhance the antibody-dependent cell-mediated cytotoxicity (ADCC) of these antibodies, thereby increasing their antitumor efficacy (Non-patent Literature 1: Kevin C. Conlon et al, JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 39, Number 1, 2019; the contents of this document are incorporated herein by reference).

[0006] IL-12 is a 70 kDa heterodimer cytokine composed of two disulfide-linked proteins, IL-12A p35 (35 kDa) and IL-12B p40 (40 kDa). It is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) and is essential for initiating an effective immune response.

[0007] IL-12 emerged as one of the most potent drugs for anti-cancer immunotherapy. Given its central role in T-cell and NK-cell-mediated inflammatory responses, localized IL-12 expression remains one of the most effective ways to overcome immunosuppression. However, the clinical application of IL-12-based therapies still faces many challenges due to the potential for lethal toxicity associated with systemic administration.

[0008] Oncolytic viruses encoding IL-12 have shown potent antitumor effects in preclinical models of cancer (Non-patent Literature 2: Pan, WY et al., Mol. Ther. 20(5), 927-937 (2012), the contents of which are incorporated herein by reference), but systemic accumulation of IL-12 after delivery by oncolytic viruses leaves potential lethality in patients. Inefficient transduction of tumor cells using carrier vectors currently limits the overall antitumor effect of this approach.

[0009] More promising drug-induced IL-12 systems are expected to allow for long-term management of IL-12 levels at low toxicity levels for clinical use. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kevin C. Conlon et. al, JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 39, Number 1, 2019 [Non-Patent Document 2] Pan, WY et al., Mol. Ther. 20(5), 927-937 (2012) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The present disclosure relates to improved cytokine immunotherapy for cancer and / or inflammatory diseases. **Means for Solving the Problems**

[0012] Specifically, the present disclosure relates to a novel immunologically active alphavirus replicon vector comprising a polynucleotide encoding alphavirus non-structural proteins nsp1, nsp2, nsp3, and nsp4 and a polypeptide comprising a cytokine. Such a vector is useful for the treatment of cancer and / or inflammatory diseases while minimizing toxicity.

[0013] In another aspect, the present disclosure provides a composition comprising the alphavirus replicon vector discussed above and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a delivery medium in which the vector is encapsulated. In a preferred embodiment, the delivery medium may be an alphavirus particle consisting of alphavirus structural proteins that may include a capsid and / or envelope protein. In another embodiment, the delivery medium may be a lipid nanoparticle (LNP).

[0014] In another aspect, the present disclosure provides the alphavirus replicon vector or composition discussed above for use in the treatment or immunization against cancer or inflammatory diseases.

[0015] In another aspect, the present disclosure provides the use of the alphavirus replicon vector or composition discussed above in the manufacture of a medicament for the treatment or immunization against cancer or inflammatory diseases.

[0016] In another aspect, the present disclosure provides a method for treating and / or immunizing against cancer or inflammatory diseases in a subject, the method comprising administering to a subject in need thereof an effective amount of the alphavirus replicon vector or composition discussed above. **Brief Description of the Drawings**

[0017] [Figure 1] Figure 1 shows a representative construct of an alphavirus replicon vector. [Figure 2] Figure 2 shows the full-length VEEV TC-83 vector of a construct containing a polynucleotide encoding the construct containing IL-12-linker-TM prepared in Example 3. [Figure 3] Figure 3 shows the full-length VEEV TC-83 vector of a construct containing a polynucleotide encoding the construct containing IL-12 prepared in Example 3. [Figure 4] Figure 4 shows the effect of an IL-12 alphavirus replicon constructed to express construct 1 on MC-38 tumor model mice. [Figure 5] Figure 5 shows the effect of an IL-12 alphavirus replicon constructed to express construct 1 on CT-26 tumor model mice. [Figure 6a] Figure 6a shows the effect of an IL-12 alphavirus replicon constructed to express construct 6 on CT-26 tumor model mice. [Figure 6b] Figure 6b shows the effect of an IL-12 alphavirus replicon constructed to express construct 6 on CT-26 tumor model mice. [Figure 7a] Figure 7a shows the effect of an IL-12 alphavirus replicon constructed to express construct 1 on macrophage M1 and M2 populations in TIL of MC-38 tumor model mice. [Figure 7b] Figure 7b shows the effect of an IL-12 alphavirus replicon constructed to express construct 1 on the Treg population in TIL of MC-38 tumor model mice. [Figure 8] Figure 8 shows the effect of an IL-12 alphavirus replicon constructed to express construct 1 on B16F10 tumor model mice. [Modes for carrying out the invention]

[0018] As used herein, “cytokines” are polypeptides / glycoproteins derived from natural lymphokines (cytokines produced by lymphocytes), monokines (cytokines produced by monocytes), chemokines (cytokines with chemotactic activity), interleukins (cytokines produced by one type of leukocyte that act on another type of leukocyte), or their variants. Modified cytokines may be fragments of natural cytokines. In one embodiment, a modified cytokine has at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% amino acid sequence homology to a natural cytokine. In one embodiment, a modified cytokine is a variant in which up to 10% of amino acids are deleted, substituted, and / or added to a natural cytokine.

[0019] Examples of cytokines include interleukins (IL), encompassing over 30 types such as IL-1α, IL-1β, IL-2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13 to -37; interferons (IFN), such as IFN-α, IFN-β, and IFN-γ; tumor necrosis factor (TNF), such as TNF-α and TNF-β; transforming growth factors (TGF), such as TGF-α and TGF-β; granulocyte colony-stimulating factor (G-CSF); and granulocyte-macrophage-colony-stimulating factor (G-CSF). Colony-stimulating factors (CSF) such as macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), stem cell factor (SCF), and monocyte chemochemoactivating factor (MCAF); epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), thrombopoietin (TPO), and bone morphogenic protein. Growth factors (GFs) such as protein (BMP); other polypeptide factors including LIF, KIT ligand (KL), MPO (myeloperoxidase), and CRP (C-reactive protein); COX (cyclooxygenases) such as COX-1, COX-2, and COX-3; NOS (nitric oxide synthases) such as NOS-1, NOS-2, and NOS-3; and their variants.

[0020] Cytokines also include chemokines, which are cytokines that induce chemotaxis. There are two main classes of chemokines, CXC and CC. Neutrophil-activating protein-2 (NAP-2) and malignant melanoma growth-stimulating cytokines. sex Protein (melanoma growth stimulatory activity) CXC chemokines such as protein (MGSA) are primarily chemotactic to neutrophils and T lymphocytes, while macrophage inflammatory proteins (MIPs) including RANTES, MIP-1α and MIP-1β, keratinocyte-derived chemokines (KCs), and monochlorophyll chemokines are chemotactic to neutrophils and T lymphocytes. ball Chemotactic proteins (monocytes) CC chemokines such as chemotactic proteins (MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5), as well as eotaxins (-1 and -2), are chemotactic, particularly to macrophages, T lymphocytes, eosinophils, neutrophils, dendritic cells, and basophils. In addition, chemokines not classified into either of the major chemokine subfamilies, lymphotactin-1, lymphotactin-2 (all C Chemokines and fractalkines (CX3C chemokines) also exist.

[0021] A preferred example of a cytokine is IL-12, but it is not limited to this.

[0022] In the alphavirus repliconvectors of this disclosure, the gene encoding the cytokine may be fused to the gene encoding the transmembrane domain. As used herein, the “transmembrane domain (TM)” is a protein derived from either a natural or synthetic source. If the source is natural, in some embodiments the domain is derived from some membrane-bound or transmembrane protein. In one embodiment, the membrane-bound or transmembrane protein is a protein different from the cytokine. Examples of membrane-bound or transmembrane proteins include the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CDDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; Toll-like receptors (TLRs) such as TLR1-TLR10 in humans and TLR1-TLR9, TLR11-TLR13 in mice; interleukin (IL) receptors such as the IL-1-28 receptor, RANTES receptors (CCR1, CCR3, CCR5), MIP-1 receptor, PF4 receptor, M-CSF receptor, and NAP-2 receptor, which belongs to the GPCR chemokine receptors; and hemagglutinin (HA).

[0023] Examples of transmembrane proteins include the following: 5-lipoxygenase-activating protein, ABC transporter, ACBP, amyloid-beta (A4), Bcl-2 inhibitor, BNIP, CAAX protease, cytochrome P450, E-NPP, EPHA1, EPHA2, EPHA3, EPHA4, fatty acid desaturase, gamma secretase, glucose transporter, glycophorin, GPCR, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, HSD-11β, hypoxia-inducible protein, immunoglobulin, insulin receptor, integrin, ion channel, MAPEG, MFS, MinK family, MPP, peptidase AD, peptidase family M48, peptidase MA jagged protein, receptor kinase, SNARE complex, sulfatase, TNF receptor, transmembrane proteins 14 14) Transporters, TROBP, VEGF receptors, aldehyde dehydrogenases, ammonia and urea transporters, FMN-binding oxidoreductases, leucine-rich repeat (LRR)-containing transmembrane proteins, leukotriene C4 synthases, lysosomal membrane glycoproteins, major endogenous membrane proteins (MIP) / FNT superfamily, microsomal prostaglandin E synthases, N-(deoxy)ribosyltransferase-like membrane proteins, neutral / alkaline ceramidases, oligosaccharide transferases, pentamer ligand-dependent ion channels, rhodopsin-like receptors and pumps, single-helix ATPase regulators, squalene / phytoene synthases, stearoyl-CoA desaturase 1, stannin (SNN) membrane proteins, T cell surface glycoprotein CD3 zeta chains, tetratricopeptide repeat (TPR) alpha-helix repeat proteins, and transmembrane proteins having NAD(P)-binding Rothman-fold domains.

[0024] Furthermore, monocellular / peripheral proteins or other intramembrane proteins and peptides bound to the lipid bilayer can also be used as transmembrane proteins. Examples include alpha / beta hydrolases, annexins, Bet V1-like proteins, C1 domain-containing proteins, C2 domain-containing proteins, CoA-dependent acyltransferases, CRAL-TRIO domain-containing proteins, DNA-degrading enzyme I-like proteins, fibrinogen, FYVE / PHD zinc finger proteins, galactose-binding domain-like proteins, glycolipid transfer proteins, immunoglobulin-like superfamily (E Set) proteins, lipokines, lipoxygenases, PGBD superfamily, PH domain-like proteins, phosphatidylinositol 3- / 4-kinases, PLC-like phosphodiesterases, phosphotyrosine protein phosphatase II, P-loop-containing nucleoside triphosphate hydrolases, protein kinase superfamily, PX domain-containing proteins, saposins, synucleins, and the transcription factor tubby.

[0025] As used in this disclosure, the term “transmembrane domain” includes at least the transmembrane region of a membrane-bound or transmembrane protein. Furthermore, the transmembrane domain may also include the near-membrane domain (JMD) and / or cytoplasmic end of a membrane-bound or transmembrane protein.

[0026] Alternatively, in some embodiments, the transmembrane domain is synthesized. In some embodiments, the synthesized transmembrane domain mainly contains hydrophobic residues such as leucine and valine. In addition, highly charged residues are adjacent to the transmembrane domain (transport stop signals). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthesized transmembrane domain.

[0027] Preferred examples of transmembrane domains may be those derived from TLR4 (Toll-like receptor 4) and influenza virus hemagglutinin (HA). Specific examples include proteins consisting of a mobile near-membrane region or mobile linker, a transmembrane domain of influenza virus hemagglutinin, and the cytoplasmic terminal of the protein "HA (mobile-TM-Cyt)", as well as proteins consisting of TLR4 (Toll-like receptor 4).

[0028] By fusing a cytokine-encoding gene with a transmembrane domain, particularly a gene encoding the influenza virus hemagglutinin transmembrane domain, the safety of the resulting alphavirus repliconvector is improved while maintaining its immunological activity-inducing effect.

[0029] The cytokine polypeptide and the transmembrane domain may be fused directly or indirectly. In one embodiment, one or two linkers may be interposed between them.

[0030] Furthermore, cytokine polypeptides and transmembrane domains can be cleaved and replaced with short linkers. In some embodiments, cytokine structural proteins and heterologous transmembrane domains include one or more peptide linkers.

[0031] An example of a short linker is one consisting of 2 to 25 amino acids (e.g., 2, 3, 4, 5, or 6 amino acids). Typically, short linkers are 2 to 15 amino acid long, such as SG, GS, SGG, GGS, SGSG, and TRGGS. In certain situations, there may be only one linker, such as glycine (G), serine (S), and cysteine ​​(C).

[0032] When cytokine polypeptides are chemically conjugated to heterologous transmembrane domains via chemical crosslinkers, examples of crosslinkers include, but are not limited to, SMPH, sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, and SIA.

[0033] IgG-derived substances can also be used as linkers. Examples of IgG-derived substances include IgG1 to IgG4, which include (i) full-length (hinge-CH2CH3), (ii) half-length (hinge-CH3), and (iii) short-chain (12aa hinge only). A preferred example is IgG4-CH3.

[0034] The construct may also include a signal sequence. As used herein, a “signal sequence” (sometimes referred to as a signal peptide, targeting signal, localization signal, localization sequence, transition peptide, leader sequence, or leader peptide) is, depending on the context, a polynucleotide or polypeptide. The signal sequence is approximately 9–200 nucleotides or 3–70 amino acids long and may be incorporated into the 5' end of a coding region or the N-terminus of a protein. Some signal sequences are cleaved from the protein by a signal peptidase, for example, after the protein has been transported to the desired position.

[0035] The signal sequence may be that of the target protein that is to be expressed by the alphavirus replicon.

[0036] As used herein, "alphavirus" refers to RNA-containing viruses belonging to the family Togaviridae. Examples of Togaviridae viruses include Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus, Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus (CHIKV), O'nyong-nyong Virus, Ross River Virus, Barma Forest Virus, and Getavirus. Examples include, but are not limited to, the following viruses: Sagiyama Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu Virus, Buggy Creek Virus, and Ockelbo Virus.

[0037] "Alphavirus structural protein" means a polypeptide or fragment thereof having at least about 80% amino acid sequence homology to a natural viral capsid or envelope protein. In one embodiment, the alphavirus structural protein has at least about 85%, 90%, 95%, or higher amino acid sequence homology to Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Evaglaze Virus, Mukambo Virus, Pixna Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semryki Forest Virus, Middleberg Virus, Chikungunya Virus (CHIKV), Onyonnyon Virus, Ross River Virus, Burma Forest Virus, Geta Virus, Sagiyama Virus, Beval Virus, Mayaro Virus, Una Virus, Aura Virus, Wataroa Virus, Babanki Virus, Kyzilagati Virus, Highlands J Virus, Fort Morgan Virus, Ndum Virus, or Buggy Creek Virus. The wild-type amino acid sequence of the alphavirus structural protein can be obtained from GenBank.

[0038] In certain embodiments, the alphavirus is CHIKV, for example, CHIKV 37997 or LR2006 OPY-1 strain. In other embodiments, the alphavirus is VEEV, for example, VEEV TC-83 strain.

[0039] An "alphavirus replicon" refers to an RNA molecule capable of inducing its own amplification in target cells in vivo. The replicon encodes a polymerase (nspl, nsp2, nsp3, nsp4) that catalyzes RNA amplification and contains cis-RNA sequences necessary for replication, which are recognized and utilized by the encoded polymerase. An alphavirus replicon typically includes elements arranged in the following order: 5'UTR, sequences encoding alphavirus non-structural proteins (nspl, nsp2, nsp3, nsp4), 3'UTR, and a poly(A) signal. The alphavirus replicon also includes promoters of one or more viral subgenomes that induce the expression of the gene of interest. These sequences may have one or more mutations taught in the prior art.

[0040] In this disclosure, “alphavirus replicon,” “alphavirus replicon vector,” and “replicon” are used to refer to the same substance.

[0041] The alphavirus replicas provided by this disclosure may have the construct shown in Figure 1.

[0042] An "alphavirus replicon particle" (ARP) refers to an alphavirus replicon packaged with alphavirus structural protein. ARPs do not contain any polynucleotides that encode alphavirus structural protein.

[0043] In this disclosure, “comprises,” “comprising,” “containing,” and “having,” etc., have the meanings assigned to them in U.S. patent law and may mean “includes,” “including,” etc.; similarly, “consisting essentially of” or “consists essentially” also have the meanings assigned to them in U.S. patent law and this term is non-restrictive, allowing for more entities than those enumerated, provided that the more entities do not alter the fundamental or novel characteristics of those enumerated, but excluding embodiments of the prior art.

[0044] A "fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. The fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0045] "Reference" means a standard or contrasting condition.

[0046] A "reference sequence" is a defined sequence used as the basis for sequence comparison. A reference sequence may be a part or all of a specified sequence; for example, it may be a full-length cDNA or a segment of a gene sequence, or a complete cDNA or gene sequence. In the case of polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. In the case of nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides, or about 300 nucleotides, or any integer around or in between.

[0047] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX program). Such software matches identical or similar sequences by attributing the degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity may involve using the BLAST program with probability scores between e<"3> and e<"100> that point to closely related sequences.

[0048] The “effective dose” refers to the amount of drug required to improve the symptoms of a disease compared to an untreated patient. The effective dose of the active compound used to carry out the present invention for the prevention or treatment of a disease varies depending on the method of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and administration plan. Such a dose is called the “effective” dose.

[0049] Good results, 10 per session 3 ~10 10 Infectious unit (IU) or 0.01 to 500 μg, preferably 10 per dose. 5 ~10 10 IU or 0.1-100 μg, for example, 10 per dose. 7 ~10 9 The alphavirus repliconvector may be administered systemically in amounts of IU or 1 to 50 μg in 1 to 8 doses, for example, by intramuscular, intratumoral, subcutaneous, or intravenous administration. The alphavirus repliconvector may preferably be formulated with a vaccine composition suitable for administration by conventional methods.

[0050] "Subjects" means mammals, including humans, or non-human mammals such as cattle, horses, dogs, sheep, or cats, but is not limited to these.

[0051] As used herein, terms such as “to treat,” “to treat,” and “treatment” refer to reducing or improving a disease and / or its associated symptoms. It will be understood, though not excluded, that treating a disorder or condition does not require the complete elimination of the disease, condition, or its associated symptoms.

[0052] As used herein, terms such as “prevent,” “prevention,” “prevention,” and “preventive measures” refer to reducing the probability of an impairment or condition occurring in an object that does not currently have such an impairment or condition but is at risk of or likely to develop one.

[0053] Unless otherwise specified or made clear from the context, the term “or” as used herein is understood to be inclusive.

[0054] Examples of treatable cancers include, but are not limited to, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, and non-small cell lung cancer. Other examples of cancer include bone cancer, pancreatic cancer, skin cancer (e.g., melanoma), cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, liver cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia This includes, but is not limited to, hematological diseases, chronic or acute leukemia including acute lymphoblastic leukemia and chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, and combinations thereof.

[0055] "Inflammatory diseases" include inflammatory conditions with autoimmune etiologies, such as arthritis [e.g., rheumatoid arthritis, chronic progressive arthritis, and osteoarthritis] and rheumatic diseases, as well as inflammatory conditions and rheumatic diseases with allergies, including osteoporosis, inflammatory pain, spondyloarthritis including ankylosing spondylitis, Reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis, and enteritis arthritis, enthesitis, hypersensitivity (including both airway hypersensitivity and dermal hypersensitivity), and allergic conditions. Specific autoimmune diseases include autoimmune hematological disorders (e.g., hemolytic anemia, aplastic anemia, pure red cell adenoma, and idiopathic thrombocytopenia), systemic lupus erythematosus (SLE), lupus nephritis, inflammatory myopathy (dermatomyositis), periodontitis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens-Johnson syndrome, and idiopathic sprue. Autoimmune inflammatory bowel disease (including ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine eye disorders, Graves' disease, sarcomatoid disease, multiple sclerosis, systemic sclerosis, fibrous diseases, primary biliary cirrhosis, juvenile diabetes mellitus (type 1 diabetes mellitus), uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, periarthritis of prostheses, glomerulonephritis (e.g., idiopathic nephrotic syndrome or Conditions that may cause uveitis include minimal change nephrotic syndrome (with or without nephrotic syndrome), multiple myeloma and other types of tumors, inflammatory diseases of the skin and cornea, myositis, loosening of bone implants, metabolic disorders (such as obesity, atherosclerosis, and other cardiovascular diseases including dilated cardiomyopathy, myocarditis, type II diabetes mellitus, and dyslipidemia), autoimmune thyroid diseases (including Hashimoto's thyroiditis), primary small and medium vasculitis, large vasculitis including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjögren's syndrome, Behçet's disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle diseases, autoimmune peripheral neuropathy, immune kidney, liver and thyroid diseases, inflammatory and atherothrombotic diseases, autoinflammatory fever syndromes, immunohematological disorders, and vesicular disorders of the skin and mucous membranes. Anatomically, uveitis can be anterior, intermediate, posterior, or panuveitis.Uveitis can be chronic or acute. The etiology of uveitis can be autoimmune, non-infectious, infectious, associated with a systemic disease, or white spot syndrome.

[0056] In any of the embodiments described above, the method further comprises administering a second composition comprising a checkpoint inhibitor polypeptide or a polynucleotide encoding it and an optional pharmaceutically acceptable carrier. In some embodiments, the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof. In some embodiments, the checkpoint inhibitor polypeptide is an antibody. In some embodiments, the checkpoint inhibitor polypeptide is an antibody selected from an anti-CTLA4 antibody or its antigen-binding fragment that specifically binds to CTLA4, an anti-PD1 antibody or its antigen-binding fragment that specifically binds to PD1, an anti-PD-L1 antibody or its antigen-binding fragment that specifically binds to PD-L1, and a combination thereof. In some embodiments, the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab. In some embodiments, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some embodiments, the checkpoint inhibitor polypeptide is an anti-PDl antibody selected from nivolumab or pembrolizumab.

[0057] Unless otherwise specified or the context makes clear, the terms “a,” “an,” and “the” as used herein shall be understood to be singular or plural.

[0058] In the art, it is acknowledged that while polynucleotide sequences described herein and in the claims are represented by "T" for typical DNA sequences, "T" is replaced by "U" when the sequence is RNA.

[0059] Any composition or method provided herein may be combined with one or more of the other compositions and methods provided herein.

[0060] The term "vector" refers to a means by which nucleic acid sequences can be replicated and / or transferred between organisms, cells, or cellular components. Examples of vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, and artificial chromosomes, which can autonomously replicate or be incorporated into the chromosomes of host cells. Vectors can also be non-self-replicating, such as naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same chain, polylysine conjugate DNA or RNA, peptide conjugate DNA or RNA, and liposomal conjugate DNA. In many, but not all, common embodiments of the present invention, the vector is a plasmid or bacmid.

[0061] Typically, nucleic acid molecules intended for expression are "operably linked" to a promoter and / or transcription factor, and are subject to transcriptional regulation by the promoter and / or transcription factor.

[0062] In one embodiment, RNA molecules such as alphavirus replicons may be generated from a template DNA sequence by conventional procedures known in the art. In vitro transcription (IVT) methods enable template-dependent synthesis of RNA molecules. IVT methods enable the large-scale synthesis of RNA transcripts. Generally, IVT utilizes a DNA template containing a promoter sequence upstream of the sequence of interest. Promoter sequences are most commonly derived from bacteriophages, such as T7, T3, or SP6 promoter sequences, but many other promoter sequences, including novelly designed ones, are acceptable. Transcription of the DNA template is typically most effectively performed by using an RNA polymerase corresponding to a specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase. IVT is generally initiated on dsDNA but can continue on a single strand. Kits for in vitro transcription, such as the T7 Transcription Kit (RiboMax® Express Large Scale RNA Production System, Promega (WI USA)).

[0063] The transfection method and the choice of expression medium will depend on the selected host system. Transfection methods are described, for example, in Ausubel et al. (above); expression mediums may be selected from those shown, for example, in Cloning Vectors: A Laboratory Manual (PH Pouwels et al., 1985, Supp. 1987). References cited in this paragraph are incorporated herein by reference.

[0064] Various expression systems exist for the preparation of the construct of the present invention. Useful expression vectors for preparing the construct include, but are not limited to, chromosome vectors, episome vectors, and virus-derived vectors. Examples include vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, alphaviruses (e.g., chikungunya virus (CHIKV) and Venezuelan encephalitis virus (VEEV)), baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses, and retroviruses, as well as vectors derived from combinations thereof.

[0065] The constructs and / or alphaviral replicon vectors used herein include alphaviral polynucleotides encoding structural proteins such as envelope proteins or capsid proteins, as described herein. The constructs and / or vectors used herein also include alphaviral polynucleotides encoding non-structural proteins nsp1, nsp2, nsp3, and nsp4 and the gene of interest. Specific examples of such constructs or vectors are shown in Figure 1.

[0066] The vector may be, for example, a phage, plasmid, virus, or retroviral vector. The construct and / or vector containing nucleotides should be operably ligated to a suitable promoter, and non-limiting examples include the CMV promoter, the phage λ PL promoter, the E. coli (E. coli) lac, phoA, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs. Other suitable promoters will be understood by those skilled in the art depending on the host cell and / or the desired expression rate. The expression construct will further contain a transcription start site, a transcription stop site, and a ribosome binding site for translation in the region to be transcribed. The coding portion of the transcript expressed by the construct will preferably contain a translation start codon appropriately placed at the beginning and a translation stop codon appropriately placed at the end of the polypeptide to be translated.

[0067] The vector will preferably contain at least one selection marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin, or ampicillin resistance genes for culture in E. coli and other bacteria. Preferred vectors include viral vectors such as baculoviruses, poxviruses (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenoviruses [e.g., canine adenovirus], herpesviruses, and retroviruses. Other vectors that may be used in the present invention include vectors for bacterial use, such as pQE70, pQE60, and pQE-9, pBluescript vector, Phagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Preferred eukaryotic vectors include pFastBacl, pWINEO, pSV2CAT, pOG44, pXTl, and pSG, pSVK3, pBPV, pMSG, and pSVL. Other preferred vectors will be readily apparent to those skilled in the art.

[0068] Recombinant constructs are prepared and can be used for transfection, and viral proteins, including the viral proteins described herein, can be expressed in eukaryotic and / or prokaryotic cells. Thus, in one embodiment, the disclosure provides a host cell comprising a vector containing nucleic acids encoding alphavirus structural proteins, including the capsid, E3, E2, 6K, and El, or portions thereof, under conditions that allow for the formation of alphavirus replicon particles, and a vector comprising nucleic acids encoding alphavirus nsp1, nsp2, nsp3, and nsp4 and at least one of the genes of interest encoding cytokines. The term “alphavirus replicon particle” refers to a particle consisting of alphavirus structural proteins enclosing an alphavirus replicon vector containing polynucleotides encoding alphavirus non-structural proteins nsp1, nsp2, nsp3, and nsp4 and polypeptides containing cytokines.

[0069] In one embodiment, the vector is a recombinant baculovirus. In another embodiment, the recombinant baculovirus is transfected into insect cells. In one preferred embodiment, the cells are insect cells. In another embodiment, the insect cells are Sf9 cells.

[0070] One notable bacterial expression system for polypeptide production is the *E. coli* pET expression system (Novagen, Inc., Madison, Wis). According to this system, the polypeptide-encoding DNA is inserted into the pET vector in an orientation designed to enable expression. Polypeptide expression is achieved by inducing the expression of T7 RNA polymerase in host cells, since the gene encoding such polypeptides is under the control of the T7 regulatory signal. This is typically achieved by using a host strain that expresses T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant polypeptide is then isolated by standard methods known in the art, e.g., the method described herein.

[0071] Depending on the selected vector and host cell, the construct is formed by growing the vector-transfected host cell under conditions in which recombinant protein is expressed and alphavirus replicons are generated, thereby forming a construct containing alphavirus replicons packaged in particles of alphavirus structural protein. In one embodiment, the present invention includes a method for producing a construct, comprising co-transfecting a vector containing polynucleotides encoding alphavirus non-structural proteins nsp1, nsp2, nsp3, and nsp4 and a target gene encoding a polypeptide containing at least one cytokine into a suitable host cell with at least one vector each encoding at least one alphavirus structural protein, and expressing the alphavirus structural proteins under conditions that enable construct formation. In another embodiment, the eukaryotic cell is selected from the group consisting of yeast, insect, amphibian, bird, or mammalian cells. The selection of appropriate growth conditions is within the scope of the art or the technician of the art.

[0072] Methods for growing cells that produce the alphavirus replicon particles of this disclosure include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. In one embodiment, cells co-transfected with a vector encoding an alphavirus replicon, such as a CHIKV or VEEV-derived vector, a vector containing a polypeptide encoding a capsid, and a vector containing a polynucleotide encoding an envelope protein are grown in a bioreactor or fermentation chamber in which the cells grow and express a protein (e.g., a recombinant protein), and then purified and isolated. Typically, cell culture is carried out under sterile, temperature, and atmosphere-controlled conditions. A bioreactor is a chamber used to culture cells in which environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a sterile plastic bag (e.g., Cellbag.RTM., Wave Biotech, Bridgewater, NJ; the contents of this reference are incorporated herein by reference). In other embodiments, the sterilized plastic bag is a bag with a capacity of approximately 50L to 1000L.

[0073] As used herein, the term “pharmaceutically acceptable carrier” means one or more miscible solid or liquid extenders, diluents, or encapsulants suitable for administration to humans or other vertebrates, and includes all aqueous solvents (e.g., water, alcoholic solutions / aqueous solutions, saline, parenteral vehicles such as sodium chloride, and ringel dextrose), non-aqueous solvents (e.g., injectable organic esters such as propylene glycol, polyethylene glycol, vegetable oil, and ethyl oleate), dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, pigments, fluids, and nutritional solutions), materials and combinations thereof that would be known to those skilled in the art. The pH and precise concentrations of individual components in a pharmaceutical composition are adjusted according to known parameters.

[0074] The encapsulating material refers to a delivery medium that packages a polynucleotide or vector, such as replicon particles (e.g., alphavirus replicon particles described in U.S. Patent Publication No. 2019-0185822, the contents of which are incorporated herein by reference) and lipid delivery systems (e.g., liposomes).

[0075] In some embodiments, the compositions or formulations of the Disclosure include a lipid delivery system, e.g., liposomes, lipoplexes, lipid nanoparticles, or any combination thereof. The alphavirus repliconvectors described herein may be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the effectiveness of polynucleotides that direct protein production, as these formulations can enhance cell transfection by polynucleotides and / or enhance the translation of encoded proteins. Liposomes, lipoplexes, or lipid nanoparticles may also be used to enhance the stability of polynucleotides.

[0076] Liposomes are artificially prepared vesicles, primarily composed of lipid bilayers, that can be used as delivery vehicles for pharmaceutical formulations. Liposomes can vary in size. Multilayer vesicles (MLVs) may have a diameter of several hundred nanometers and may contain a continuous concentric bilayer separated by narrow aqueous compartments. Small monolayer vesicles (undetermined) (SUVs) may have a diameter of less than 50 nm, and large monolayer vesicles (LUVs) may have a diameter between 50 and 500 nm. Liposome designs may include, but are not limited to, opsonins or ligands to improve liposome adhesion to unhealthy tissues, or to activate events such as endocytosis. Liposomes may maintain low or high pH values ​​to improve the delivery of pharmaceutical formulations.

[0077] Liposome formation may depend on the pharmaceutical formulation and liposomal components to be encapsulated, the properties of the vehicle in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any additional processes required during vesicle application and / or delivery, the optimal vesicle size, polydispersity, and shelf life for the intended application, as well as batch-to-batch reproducibility and scale-up production of safe and effective liposome products.

[0078] In some embodiments, the alphavirus repliconvectors described herein may be encapsulated by liposomes and / or contained in an aqueous core that is subsequently encapsulated by liposomes.

[0079] In some embodiments, the alphavirus replicon vectors described herein can be formulated as cationic oil-in-water emulsions, in which emulsion particles contain an oily core and cationic lipids that can interact with polynucleotides immobilizing molecules to the emulsion particles. In some embodiments, the vectors described herein can be formulated as water-in-oil emulsions comprising a continuous hydrophobic phase in which a hydrophilic phase is dispersed.

[0080] In some embodiments, the alphavirus repliconvectors described herein can be formulated as lipid-polycation complexes. Non-limiting examples include the polycation being a cationic peptide, or, but not limited to, a polypeptide such as polylysine, polyornithine, and / or polyarginine, and a cationic peptide.

[0081] In some embodiments, the alphavirus repliconvectors described herein can be formulated as lipid nanoparticles (LNPs).

[0082] Lipid nanoparticle formulations typically contain one or more lipids. In some embodiments, the lipids are cationic or ionizable lipids. In some embodiments, the lipid nanoparticle formulations further contain other components, such as PEG or PEG-modified lipids, including phospholipids, structural lipids, quaternary amine compounds, and molecules that can reduce particle aggregation. In some embodiments, the amount of cationic and ionizable lipids in the lipid composition ranges from about 0.01 mol% to about 99 mol%.

[0083] LNPs contain pH-sensitive, ionizable cationic lipids that attract anionic nucleic acids to form a self-assembling nanoparticle core, ensuring a high degree of encapsulation. At physiological pH, LNPs are neutral, eliminating the toxic mechanisms found in molecules that are permanently cationic.

[0084] These similar pH-sensitive lipids respond to the acidic environment of endosomes, inducing endosome disruption and the release of nucleic acids into the cell.

[0085] This replicon-based technology is a unique platform technology for vaccination because RNA can self-amplify to produce vaccine antigens and deliver them into cellular organelles. Furthermore, this replicon-based technology overcomes the challenges commonly associated with DNA-based vaccines, such as the risks and equipment associated with genomic integration or high doses required for administration (e.g., electroporation), and is expected to offer superior immunogenicity compared to mRNA technology at minimum doses based on a self-replicating system.

[0086] According to this disclosure, novel immunologically active alphavirus repliconvectors containing nucleic acids encoding cytokine proteins / polypeptides are useful for treating cancer and / or inflammatory diseases while minimizing toxicity.

[0087] The present invention will be described in detail with reference to the following embodiments. However, these embodiments are not intended to limit the scope of this application. [Examples]

[0088] [Example 1]

[0089] The genes encoding constructs 1-6 shown below were synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages).

[0090] Construct 1: The gene encoding the mouse IL-12 sequence shown below.

[0091] [ka] MACPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTC GMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS VPGVGVPGVG RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(Sequence ID 1) Linker is underlined.

[0092] Mouse IL-12B (p40) MACPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS(Sequence ID 2)

[0093] Mouse IL-12A (35p) without signal sequence RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA(Sequence ID 3) Linker VPGVGVPGVG (Sequence ID 4)

[0094] In the following constructs 2-6, "mouse IL-12 (p40-p35)" corresponds to construct 1.

[0095] Construct 2: A gene encoding mouse IL-12 (p40-p35), fused to human IgG4CH3 and HA (mobile domain-transmembrane (TM)-cytoplasmic terminal (Cyt)) using a linker.

[0096] [ka] MACPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNY SGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSL KFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSVPGVGVPGVGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKT CLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA GS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GS GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(Sequence ID 5) The underlined "GS" stands for linker.

[0097] Human IgG4 CH3: GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 6) GS: Linker (GGATCC) HA (mobility-TM-Cyt): GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(Sequence ID 7)

[0098] Construct 3: The gene encoding mouse IL-12 (p40-p35) was fused to HA (mobility-TM-Cyt) using a linker.

[0099] [ka] MACPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNY SGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSL KFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSVPGVGVPGVGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKT CLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA GS GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(Sequence ID 8) The underlined "GS" stands for linker.

[0100] Construct 4: A gene encoding mouse IL-12 (p40-p35) fused to human IgG4CH3 and human TLR4 (TM-Toll / interleukin-1 receptor domain (TIR)) using a linker.

[0101] [ka] MACPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNY SGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSL KFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSVPGVGVPGVGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKT CLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA GS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (Sequence ID 9) The underlined "GS" stands for linker.

[0102] Human TLR4 (TM-TIR): KTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIK YG RGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (Sequence ID 10)

[0103] Construct 5: A gene encoding mouse IL-12 (p40-p35) fused to human TLR4 (TM-TIR) using a linker.

[0104] [ka] MACPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNY SGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSL KFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSVPGVGVPGVGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKT CLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA GS KTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (Sequence ID 11) The underlined "GS" stands for linker.

[0105] Construct 6: A gene encoding mouse IL-12 (p40-p35), fused to mouse IgG4CH3 and HA (mobile domain-transmembrane (TM)-cytoplasmic terminal (Cyt)) using a linker.

[0106] [ka] MACPQKLTISWFAIVLLVSPLAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNY SGRFTCSWLVQRNMDLKFNIKSSSSPPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSL KFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSVPGVGVPGVGRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKT CLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA GS GRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK GS GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(Sequence ID 12) The underlined "GS" stands for linker.

[0107] Mouse IgG4CH3 GRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG(Sequence ID 13)

[0108] HA (mobility-TM-Cyt) and IgG4 are also disclosed as follows: HA (mobility-TM-Cyt) https: / / www.ncbi.nlm.nih.gov / protein / P03452 UniProtKB / Swiss-Prot:P03452.2 Human IgG4 https: / / www.uniprot.org / uniprot / P01861 UniProtKB:P01861 [Example 2]

[0109] The genes encoding constructs 7 and 8 were prepared. Construct 7: The following is a human IL-12 sequence.

[0110] [ka] MCHQQLVISWFSLVFLASPLVA IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS VPGVGVPGVGRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Sequence ID 14) The first underlined line represents the signal sequence (SS) of human IL-12B(p40), and the second underlined line represents the linker.

[0111] Human IL-12B (p40) MCHQQLVISWFSLVFLASPLVA IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Sequence ID 15) The underlined portion is the signal sequence (SS) of human IL-12B(p40). Linker (underlined) VPGVGVPGVG (Sequence ID 4) Human IL-12A(p35) w / o signal sequence (The signal sequence "MCPARSLLLVATLVLLDHLSLA (sequence number 16)" is missing.) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Sequence ID 17)

[0112] Construct 8: A gene encoding human IL-12 (p40-p35), fused to human IgG4CH3 and HA (mobile domain-transmembrane (TM)-cytoplasmic end (Cyt)) using a linker.

[0113] [ka] In the structure described above, "Human IL-12 (p40-p35)" corresponds to construct 7. MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEA KNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSVPGVGVPGVGRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEAC LPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS GS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GS GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (Sequence ID 18) The underlined "GS" stands for linker.

[0114] Human IL-12B (p40) UniProt:P29460 https: / / www.uniprot.org / uniprot / P29460 Human IL-12A (p35) Uniprot:P29459 https: / / www.uniprot.org / uniprot / P29459 [Example 3]

[0115] Vector preparation Figure 1 shows a diagrammatic construct of the alphavirus replicon.

[0116] Constructs 1-6 prepared in Example 1 and constructs 7 and 8 prepared in Example 2 were used as target genes, respectively. The nucleotides encoding the constructs were cloned into a VEEV replicon vector under the control of the SG promoter. By inserting the AscI and SbfI restriction sites, VEEV replicon plasmids encoding each fragment were created to obtain the full-length VEEV TC-83 replicon construct. Refer to Figure 2 for constructs containing TM, and Figure 3 for constructs without TM.

[0117] The nucleotide sequences of the SG promoter, 5'UTR, 3'UTR, and poly(A) tail are as follows. The RNA sequence was obtained by using these DNA sequences as a template. SG promoter:cctgaatggactacgacatagtctagtccgccaag (SEQ ID NO: 19) 5'UTR:ataggcggcgcatgagagaagcccagaccaattacctacccaaa(Sequence ID 20) 3UTR: gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttc (SEQ ID NO: 21) POAtail:a

[0118] The amino acid sequence of VEEV TC-83 replicon nsP1-4 is as follows: APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADV AIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERV QRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPPVEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGSPSVSSSSWSIPHAS DFDVDSLSILDTLDGASVTSGAVSAETNSYFARSMEFRARPVPAPRTVFRNPPHPAPRTRTPPLAHSRASSRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (Sequence ID 23) The amino acid sequence corresponding to nsp3 is underlined.

[0119] In this embodiment, the amino acid sequence of nsp3 corresponding to sequences 1330-1886 in SEQ ID NO: 23 was replaced with the sequence shown below. The underlined sequence differs from that of SEQ ID NO: 23. APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDR LTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILG K SMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA(Sequence ID 24) [Example 4]

[0120] Preparation of alphavirus replicon particles (ARP) HEK293T cells were transfected with 10 μg of full-length replicon plasmid, 1 μg of VEEV Env expression plasmid, and 1 μg of VEEV capsid NLS variant (or 1 μg of VEEV capsid expression plasmid) for each of the constructs 1-8 prepared in Example 3. The supernatant was collected 48-96 hours after transfection. Replicon particles were purified using an ion-exchange column. HEK293T or Vero cells were infected with the purified particles and the infectivity titer was determined. The purified replicon particles were used for therapeutic treatment. [Example 5]

[0121] Effect of an alphavirus replicon constructed to express construct 1 The IL-12 alphavirus replicon particles prepared in Example 4, constructed to express construct 1, were evaluated. For this evaluation, a mouse MC-38 subcutaneous syngeneic transplantation model was used. MC-38 is a cell line derived from C57BL6 mouse colon adenocarcinoma cells. Ten 6 cells were subcutaneously injected into the abdomen of 48 C57BL / 6 female mice. When the average tumor volume reached 75-125 mm 3 , all the mice were randomly assigned to 6 groups (n = 8 per group). Administration was started within 24 hours of randomization (day 0). Animals were administered according to Table 1 below. Replicon groups 3 and 4 evaluated a control vector constructed to express GFP as the gene of interest, and groups 5 and 6 were constructed to express mouse IL-12 (construct 1). Mice were injected intratumorally (i.t.) with the designated replicon at a dose of 1 × 10 9 infection unit dose (IU) every other day (for a total of 8 times), or in combination with 10 mg / kg of anti-mouse PD-1 monoclonal antibody (clone RMP1-14, aPD-1 mAb) by intraperitoneal injection (IP) twice a week for a total of 6 times. Animals were monitored and tumors were measured twice a week during the study period. The results are shown in Figure 3. Figure 3 shows the mean and error (s.e.m.) of tumor size in each group.

[0122] [Table 1] The results are shown in Figure 4.

[0123] The data represent that the IL-12 alphavirus replicon and the combination of the IL-12 alphavirus replicon and anti-PD-1 antibody showed superior antitumor effects over control and anti-PD-1 monotherapy. [Example 6]

[0124] Effect of an alphavirus replicon constructed to express construct 1 IL-12 alphavirus replicon particles (ARPs) prepared in Example 4, constructed to express construct 1, were evaluated using another cancer cell line. In this example, a mouse CT-26 subcutaneous syngeneic transplant mouse model was used. CT-26 is an N-nitroso-N-carbamate methyl (NNMU) induced undifferentiated colon cancer cell line. 5 × 10⁶ particles were transplanted into the flanks of Balb / c female mice (48 mice). 5 Individual cells were injected subcutaneously. The average tumor volume was 75-125 mm². 3 Upon reaching this point, all mice were randomly assigned to one of six groups (n=8 per group). Administration was initiated within 24 hours of randomization (day 0). The animals were administered according to Table 2. Alphavirus replicon particle groups 3 and 4 were constructed to express GFP as a control vector, and groups 5 and 6 were constructed to express mouse IL-12 (Construct 1). Mice were given 4 × 10⁶ doses. 8 Specified replicons for infection units (IU) were injected intratumorally (it) every other day (a total of 8 injections), or 10 mg / kg of anti-mouse PD-1 monoclonal antibody (clone RMP1-14, aPD-1 mAb) was administered intraperitoneally (IP) twice a week for a total of 6 injections. Animals were monitored, and tumors were measured twice a week during the study period. The results are shown in Figure 5. Figure 5 shows the mean tumor size and the standard error (sem) of the mean for each group.

[0125] [Table 2]

[0126] The data indicate that IL-12 alphavirus replicons and combinations of IL-12 alphavirus replicons with anti-PD-1 antibodies demonstrated superior antitumor efficacy compared to the control and anti-PD-1 monotherapy. [Example 7]

[0127] Effect of alphavirus replicas constructed to express construct 6 We evaluated the IL-12 alphavirus replicon particles prepared in Example 4, which were constructed to express construct 6. 1.75 × 10⁶ particles were placed in the flank of Balb / c female mice. 8 Individual CT-26 cells were subcutaneously injected. The average tumor volume was 50-100 mM. 3 At this point, all mice were randomly assigned to two groups (n=8 per group). Administration was started within 24 hours of randomization (day 0). The animals were immunized with alphavirus replicon particles prepared in Example 4, expressing construct 6. Mice were given 4 × 10⁶ doses. 8 Specified replicons of infection units (IU) were injected intratumorally (it) every other day (a total of 8 injections). Animals were monitored, and tumors were measured twice a week during the study period. The results are shown in Figure 6a. Figure 6a shows the mean tumor size and standard error (sem) of the mean for each group. Figure 6b shows the tumor size for each mouse (left, vehicle; right, IL-12_HA). The cross symbols indicate that one mouse would have been sacrificed due to a large tumor size.

[0128] The data indicates that the IL-12 alphavirus replicon showed a potent antitumor effect. [Example 8]

[0129] Effect of alphavirus replicas constructed to express construct 1 IL-12 alphavirus replicon particles prepared in Example 4, constructed to express construct 1, were evaluated. MC-38 cells were subcutaneously injected into the flanks of C57BL6 female mice. The mean tumor volume was 50–100 mM. 3 Upon reaching this stage, all mice were randomly assigned to one of four groups (n=8 per group). Administration was initiated within 24 hours of randomization (day 0). Alphavirus replicon particles were constructed to express GFP as a control vector or to express mouse IL-12 (Construct 1). Mice were given 2.1 × 10⁶ doses. 8Specified infection units (IU) replicons were injected intratumorally (it) every other day on days 0, 2, 4, and 6 (a total of 4 injections), or 10 mg / kg of anti-mouse PD-1 monoclonal antibody (clone RMP1-14, aPD-1 mAb) was administered intraperitoneally (IP) on days 0 and 6. Animals were sacrificed on day 9, tumors were collected, and tumor-infiltrating lymphocytes (TILs) were analyzed by FACS. The results are shown in Figure 7. Figure 7a shows the M1 and M2 macrophage populations of macrophages contained in TILs. CD45 in living TIL cells. + CD11b + F4 / 80 + Among the cells, the M1 and M2 populations were identified using the CD206 and IA / EA markers. Figure 7b shows the CD4 contained in TIL. + This shows the proportion of regulatory T cells (Tregs) in the T cell population. TIL CD45 + CD3 + CD4 + Among the cells, FoxP3 + The proportion of cells was determined as the Treg population. Mice immunized with a mouse IL-12 expressing replicon showed that 1) the population of M1 macrophages was much higher than the population of M2 macrophages, and 2) the population of Treg cells in TILs was reduced.

[0130] While not intended to be a theoretical constraint, the inventors believe that, in addition to other known effects of IL-12, alphavirus replicons constructed to express IL-12 can provide potent antitumor effects by increasing the M1 macrophage population compared to M2 macrophages and by reducing the Treg population in TILs. [Example 9]

[0131] Effect of alphavirus replicas constructed to express construct 1 B16F10 cells were subcutaneously injected into the flanks of female C57BL6 mice. The average tumor volume was 50-100 mM. 3At the point when the mice reached 6, all mice were randomly assigned to a group (n=8 per group). B16F10 is a mouse cell line derived from melanoma. Dosage was initiated within 24 hours of randomization (day 0). Alphavirus replicon particles were constructed to express GFP as a control vector or to express mouse IL-12 (Construct 1). Mice were given 1 × 10⁶ doses. 9 Specified replicons of infection units (IU) were injected intratumorally (it) every other day (total of 8 injections), or 10 mg / kg of anti-mouse PD-1 monoclonal antibody (clone RMP1-14, aPD-1 mAb) was administered intraperitoneally (IP) (total of 6 injections). Animals were monitored, and tumors were measured twice a week during the study period. The results are shown in Figure 8. Figure 8 shows the mean tumor size and the standard error (sem) of the mean for each group.

[0132] The data indicate that IL-12 alphavirus replicons and combinations of IL-12 alphavirus replicons with anti-PD-1 antibodies demonstrated superior antitumor efficacy compared to the control and anti-PD-1 monotherapy.

Claims

1. An alphavirus replicaconvector comprising polynucleotides encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3, and nsp4 and cytokine-containing polypeptides.

2. The alphavirus replicaconvector according to claim 1, wherein the cytokine is a lymphokine, monokine, chemokine, or interleukin.

3. The alphavirus replicaconvector according to claim 1, wherein the cytokine is an interleukin.

4. The alphavirus replicaconvector according to claim 3, wherein the interleukin is interleukin-12 (IL-12).

5. An alphavirus replicaconvector according to any one of claims 1 to 4, wherein a cytokine is fused to a transmembrane domain.

6. The alphavirus replicaconvector according to claim 5, wherein the transmembrane domain is derived from influenza hemagglutinin (HA) or TLR4.

7. The alphavirus replica vector according to claim 5 or 6, wherein the transmembrane domain is fused to a cytokine by a linker.

8. The alphavirus replica vector according to claim 7, wherein the linker is IgG4CH3.

9. An alphavirus replicaconvector according to any one of claims 1 to 8, comprising a promoter, a 5'UTR, polynucleotides encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3, and nsp4, an SG promoter, a target gene encoding a cytokine-containing polypeptide, a 3'UTR, and a poly A tail.

10. The alphavirus replicaconvector according to any one of claims 1 to 9, wherein the polypeptide containing cytokines has the amino acid sequence of SEQ ID NO: 14 or 18.

11. The alphavirus replica vector according to any one of claims 1 to 10, wherein the alphavirus is CHIKV or VEEV.

12. The alphavirus replicon vector according to claim 11, wherein CHIKV is CHIKV strain 37997 or OPY-1 strain.

13. The alphavirus replicaconvector according to claim 12, wherein VEEV is the VEEV TC-83 strain.

14. (i) a pharmaceutically acceptable carrier, and (ii) The vector according to any one of claims 1 to 13 A composition containing the following:

15. The composition according to claim 14, wherein a pharmaceutically acceptable carrier is a delivery medium, and an alphavirus replicon vector is encapsulated in the delivery medium.

16. The composition according to claim 15, wherein the delivery medium is particles made of alphavirus structural proteins or a lipid delivery system.

17. The composition according to claim 16, wherein the alphavirus structural protein comprises a capsid and at least one envelope protein, and the capsid has one or more modifications in its nuclear localization signal (NLS).

18. The particle according to claim 16, wherein the alphavirus structural protein comprises at least one envelope protein E3 having one or more modifications to the furin site within the E3 protein.

19. A composition comprising lipid nanoparticles and an alphavirus replicon vector according to any one of claims 1 to 13, wherein the alphavirus replicon vector is encapsulated within the lipid nanoparticles.

20. A vaccine comprising an alphavirus replicon vector, particle, or composition according to any one of claims 1 to 19.

21. A method for treating and / or immunizing cancer or inflammatory disease in a subject, comprising administering an effective amount of the alphavirus repliconvector or composition described in any one of claims 1 to 18 to a subject in need thereof.

22. Use of the alphavirus repliconvector or composition according to any one of claims 1 to 18 for the manufacture of a pharmaceutical product for the treatment and / or immunity of cancer or inflammatory diseases.

23. An alphavirus replicaconvector or composition according to any one of claims 1 to 18, for use in the treatment and / or immunotherapy for cancer or inflammatory diseases.

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