Antigen presentation enhancer

Abstract

The goal is to enhance the presentation of antigen peptides expressed on the cell surface by binding to MHC class I molecules. [Solution] We provide an agent that enhances the amount of endogenous peptide-derived antigens presented to MHC class I molecules expressed on the cell surface, comprising a protein synthesis inhibitor.

Description

[Technical Field]

[0001] The present invention relates to an antigen presentation enhancer and a method for producing antigen presentation enhancer cells. [Background technology]

[0002] Living organisms constantly maintain homeostasis by eliminating non-self elements such as bacteria and viruses that invade their bodies, or cells that have become non-self, such as cancer cells that develop within the body. In cell-mediated immunity, which directly targets and destroys non-self cells such as cancer cells and virus-infected cells, self and non-self are distinguished based on the type of antigen peptide bound to the MHC class I molecule. MHC class I molecules are expressed on the cell surface of all nucleated cells after binding to peptides generated by the degradation of endogenous proteins in the ubiquitin-proteasome system. Antigen peptides presented on MHC class I molecules function as markers that indicate to cell-mediated immunity that a cell is self by revealing to the immune system the types and amounts of all proteins synthesized within the cell. This presentation of peptides derived from endogenous proteins to MHC class I molecules is collectively called direct presentation (DP) (Non-Patent Literature 1).

[0003] Cancer cells and virus-infected cells express cancer antigens or viral antigens derived from viruses that are not present in normal cells due to carcinogenesis, etc. Therefore, these cells present antigen peptides derived from these non-self antigens on MHC class I molecules. Cytotoxic T cells recognize cells that present such non-self antigen peptides on MHC class I molecules as non-self and induce apoptosis to eliminate the non-self cells (Non-Patent Literature 1).

[0004] MHC class I molecules bind to 8-11 amino acid oligopeptides (antigen peptides) produced by the degradation of intracellular proteins by the ubiquitin-proteasome system within the endoplasmic reticulum and are expressed on the cell surface (Non-Patent Literature 2). Here, the type and amount of antigen peptides presented on the cell surface are thought to be roughly proportional to the expression level of intracellular proteins and the degradation rate by the ubiquitin-proteasome system. Therefore, enhancing the presentation of cancer antigen peptides and viral antigen peptides on MHC class I molecules by some means is an extremely important issue in immunotherapy that utilizes cellular immunity.

[0005] Therapies that activate cellular immunity by targeting cancer antigen peptides presented on MHC class I molecules are attracting attention as science-based therapies and are being developed as cancer peptide vaccine therapy. Cancer peptide vaccine therapy is a method that identifies cancer antigen peptides specific to cancer cells and administers the cancer antigen peptides together with an immune-activating adjuvant to activate cancer cell-specific CTLs and eliminate cancer cells. In this method, it is important to accurately predict the cancer antigen peptide. However, since cancer cells originate from the body's own cells, cancer-specific proteins are also encoded in the body's own genes and are often expressed on normal cells as well. Therefore, if antigen peptides derived from self-proteins strongly bind to MHC class I molecules as cancer antigen peptides and are expressed on the surface of self-cells, it can cause serious autoimmune diseases. Cytotoxic T cells that recognize such antigen peptides that strongly bind to MHC class I molecules are likely to be eliminated during the thymic education process and therefore do not exist. For this reason, cancer antigen peptides used in cancer immunotherapy often use candidates with moderate binding ability to MHC class I molecules. However, peptides with moderate binding ability to MHC class I molecules face a problem: they compete with peptides with strong binding ability, resulting in a reduced amount being presented to MHC class I molecules. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Janeway, C. et al., 2001, The Immune system in Health and Disease 5th edn. Garland Press, New York. [Non-Patent Document 2] Yewdell, JW, et al., 2003, Nat. Rev. Immunol. 3, 952-961 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to enhance the presentation level of specific antigen peptides expressed on the cell surface by binding to MHC class I molecules. [Means for solving the problem]

[0008] To solve the above problems, the inventors focused on the source of antigen peptides. Conventionally, the source of antigen peptides has been considered to be mainly proteins that have reached the end of their lifespan and been degraded. However, in recent years, it has been thought that they may rather originate from DRiPs, which are rapidly degraded because they cannot assume a normal three-dimensional structure during the protein synthesis process.

[0009] Therefore, in order to confirm that the main source of antigen peptides is derived from DRiPs, the inventors investigated the existence of differences in the presentation efficiency of antigen peptides. Quantitative analysis of the correlation between the expression level of the antigen protein and the position of the antigen peptide derived from that antigen protein on the antigen protein yielded a novel finding: even for the same T cell antigen epitope, the N-terminal antigen epitope is presented more efficiently on the MHC class I molecule compared to the C-terminal antigen epitope. Based on this finding, the inventors succeeded in increasing the proportion of DRiPs by inhibiting the translation process of the target antigen protein, thereby enhancing the presentation efficiency of a specific antigen peptide on the MHC class I molecule, and thus completed the present invention. This specification provides the following inventions.

[0010] (1) An agent comprising a protein synthesis inhibitor that enhances the amount of endogenous peptide-derived antigens presented to MHC class I molecules expressed on the cell surface. (2) The antigen presentation enhancer described in (1), wherein the endogenous peptide is derived from DRiPs (defective ribosomal products). (3) The antigen presentation enhancer according to (2), wherein the DRiPs is tDRiPs. (4) The antigen presentation enhancer according to any one of (1) to (3), wherein the protein synthesis inhibitor comprises a low molecular weight compound, nucleic acid, or polypeptide. (5) The antigen presentation enhancer according to (4), wherein the low molecular weight compound is a substance that has peptide elongation inhibitory activity in ribosomes. (6) The antigen presentation enhancer according to any one of (1) to (5), wherein the cells are cancer cells or virus-infected cells. A composition that enhances the amount of antigen presented to MHC class I molecules by endogenous peptides in cells, comprising an antigen presentation enhancer described in any one or more of (7)(1) to (8). (8) The antigen presentation enhancement composition according to (7), comprising an expression vector containing a polynucleotide encoding a target antigen peptide in an expressible state. (9) The antigen presentation enhancement composition according to (8), wherein the target antigen peptide is a cancer cell-specific protein or a virus-specific protein, or a peptide fragment of the N-terminal region thereof. (10) The antigen presentation enhancement composition according to (8) or (9), wherein a polypeptide degradation signal is linked to the terminus of the target antigen peptide. A cellular immune-stimulating composition containing an antigen presentation-enhancing composition described in any of (11), (7), to (10) as an active ingredient. (12) A method for producing cells in which the antigen presentation amount of a target antigen peptide is enhanced in vitro, comprising an introduction step of introducing an expression vector containing a polynucleotide encoding the target antigen peptide in an expressible state into the cells, and an administration step of administering an antigen presentation enhancer according to any one of (1) to (6) to the transformed cells obtained in the introduction step. (13) The manufacturing method according to (12), wherein the cells are cells taken from a human. (14) The method for producing the target antigen peptide according to (12) or (13), wherein the target antigen peptide is a cancer cell-specific protein or a virus-specific protein, or a peptide fragment of the N-terminal region thereof. (15)(12)~(14) Antigen presentation-enhancing cells obtained by any of the manufacturing methods described. [Effects of the Invention]

[0011] According to the antigen presentation enhancer of the present invention, it is possible to enhance the presentation amount of the target antigen peptide that is expressed on the cell surface by binding to MHC class I molecules. According to the method for producing cells with enhanced antigen presentation according to the present invention, it is possible to produce cells in which the amount of a specific antigen peptide of interest presented on the cell surface is enhanced. [Brief explanation of the drawing]

[0012] [Figure 1-1] A schematic diagram of the fluorescent protein fusion antigen protein used in this example is shown. [Figure 1-2]a is a diagram quantifying and comparing the antigen presentation levels when YFP, pOV8-YFP, and YFP-pOV8 are used as antigen proteins. b is a diagram quantifying the antigen presentation level of an antigen peptide in which pOV8 in a is replaced with V-OVA. c is a diagram quantifying the antigen presentation level of an antigen peptide in which pOV8 in a is replaced with R4. d is a diagram quantifying the antigen presentation level of an antigen peptide in which YFP in a is replaced with AZ. e is a diagram quantifying and comparing the antigen presentation levels when YFP, YFP-YFP, pOV8-YFP-YFP, YFP-pOV8-YFP, and YFP-YFP-pOV8 are used as antigen proteins. All of the diagrams a to e show relative values with the MFI value of EL4-Luc2 cells expressing only a fluorescent protein (YFP or AZ) as the antigen protein set to 1. [Figure 2-1] This is a diagram showing the ratio of cells that underwent antigen-specific cell death when cytotoxic T cells purified from OT-I mice immunized with pOV8 were co-cultured with three types of EL4-Luc2 cells each stably expressing the antigen peptide of YFP, pOV8-YFP, or YFP-pOV8 at each ET ratio. In the diagram, "NS" indicates Not Significant, and "**" indicates p<0.01. [Figure 2-2] This is a diagram showing the proliferation count of transplanted EL4-Luc2 cells in individual mice, represented by the luminescence intensity of the cells, 2 weeks (Day 14) after the day of immunization with pOV8 (Day 0), following transplantation of the same number of each of the three types of EL4-Luc2 cells into OT-I mice. In the diagram, "NS" indicates Not Significant, and "**" indicates p<0.01. [Figure 2-3] This is a diagram showing the survival rate of OT-I mice immunized with pOV8 after transplanting the same number of each of the three types of EL4-Luc2 cells into the OT-I mice. [Figure 3-1]This is a diagram in which the antigen presentation level is quantified and compared, with the introduced protein amount of Chariots+YFP defined as 1, when mature proteins (YFP, pOV8-YFP, and YFP-pOV8) are transfected into EL4-Luc2 cells. In the diagram, Control indicates EL4-Luc2 cells not supplemented with mature proteins. Chariots (registered trademark: Active Motif, Inc.) indicates a reagent for introducing proteins into cells, and MG132 indicates a proteasome inhibitor. In the diagram, "NS" means Not Significant, and "**" indicates p<0.01. [Figure 3-2] This is a diagram showing the relative introduced protein amount, with the introduced protein amount of Chariots+YFP defined as 1, when the fluorescent protein introduced into cells is measured by Western blotting. In the diagram, "NS" means Not Significant. [Figure 4-1] The antigen presentation level on the cell surface in the presence of various inhibitors is shown as a relative value when the MFI value of EL4-Luc2 cells expressing only YFP is defined as 1. In the diagram, "α-Am." indicates α-amanitin, "Ani." indicates anisomycin, "Cana." indicates canavanine, "Chlo." indicates chloroquine, "CHX" indicates cycloheximide, and "Eme." indicates emetine. Further, in the diagram, "***" indicates p<0.001. [Figure 4-2] This is a diagram showing the ratio of the antigen presentation levels of pOV8-YFP and YFP-pOV8 on the cell surface in the presence of various inhibitors. In the diagram, "***" indicates p<0.001. [Figure 5-1] This is a diagram showing the relationship between the antigen presentation levels of pOV8-YFP and YFP-pOV8 by expression inhibition using RNAi for alanyl-tRNA synthetase. In this diagram, the levels are shown as relative values when the MFI value of EL4-Luc2 cells expressing only YFP is defined as 1. In the diagram, Scramble Oligo indicates an oligonucleotide for RNAi control, and ARS RNAi indicates alanyl-tRNA synthetase siRNA. In the diagram, "***" indicates p<0.001. [Figure 5-2]Figure 5-1 shows the ratio of ARS RNAi to Scramble oligo antigen presentation. [Figure 6-1] This figure shows the enhancement of specific CTL activity by protein synthesis inhibitors. It also shows the percentage of cells that underwent antigen-specific cell death when cytotoxic T cells purified from OT-I mice immunized with pOV8 were co-cultured with three types of EL4-Luc2 cells stably expressing YFP, pOV8-YFP, or YFP-pOV8 antigen peptides, respectively, at an ET ratio of 5:1, and each protein synthesis inhibitor induced antigen-specific cell death. In the figure, "NS" indicates Not Significant, and "***" indicates p<0.001. [Figure 6-2] This figure shows the enhancement of specific CTL activity by protein synthesis inhibitors. The figure also shows the proliferation rate in mice treated with each protein synthesis inhibitor after transplanting equal numbers of EL4-Luc2 cells stably expressing pOV8-YFP into OT-I mice, as indicated by the luminescence intensity of the cells. [Modes for carrying out the invention]

[0013] 1. Antigen presentation enhancers 1-1. Overview A first aspect of the present invention relates to an antigen presentation enhancer derived from endogenous peptides to MHC class I molecules expressed on the cell surface (often referred to herein as "antigen presentation enhancer"). The antigen presentation enhancer of the present invention is composed of a protein synthesis inhibitor. The antigen presentation enhancer of the present invention can increase the presentation of endogenous peptides, particularly specific antigen peptides derived from DRiPs, to MHC class I molecules.

[0014] 1-2. Definitions of Terms The following terms used in this specification are defined below. In this specification, "enhancement of antigen presentation" means increasing the amount of a specific antigen peptide presented on the cell surface beyond the normal presentation level on the cell surface.

[0015] In this specification, "antigen presentation enhancer" refers to an agent that, when administered to cells, can enhance the presentation of a specific antigen peptide on the cell surface.

[0016] In this specification, "MHC class I molecule" refers to one of the major classes of molecules in the Major Histocompatibility Complex. MHC class I molecules have the function of binding to peptide fragments produced when proteins are degraded by the ubiquitin-proteasome system within cells, expressing these peptide fragments as antigen peptides on the cell surface, and presenting them to cytotoxic T cells. This allows all nucleated cells to present their own proteins synthesized within the cell to the immune system. If a non-self antigen is presented, it is recognized as an infected cell or cancer cell, and a rapid immune response is initiated.

[0017] In this specification, "endogenous peptide" means a protein synthesized from a gene within the target cell, or a peptide fragment derived therefrom. This includes not only proteins encoded by genes contained in chromosomes within the cell, but also proteins encoded by externally introduced polynucleotides and synthesized within the target cell. The origin of the endogenous peptide is not limited. For example, it may be a peptide fragment derived from a mature protein that has (or had) functionality, or a peptide fragment derived from DRiPs.

[0018] In this specification, "DRiPs" (Defective Ribosomal Products) refers to defective polypeptides that arise during the translation process of mRNA on ribosomes due to various causes such as incorrect amino acid incorporation, premature termination of translation, translation of deleted mRNA, or abnormal three-dimensional structure due to incorrect folding. Normally, DRiPs are rapidly fragmented by the ubiquitin-proteasome system. Two types of DRiPs are known: mDRiPs and tDRiPs. "mDRiPs" (mature DRiPs) refer to proteins in which the ORF has been fully translated, or proteins that failed to form a normal three-dimensional structure. mDRiPs are rapidly fragmented by degradation by the ubiquitin-proteasome system, and equal amounts of C-terminal truncated peptides derived from the N-terminal region and N-terminal truncated peptides derived from the C-terminal region are produced from mDRiPs. "tDRiPs" (translational DRiPs) refer to proteins that have terminated prematurely while translation is incomplete. tDRiPs are also rapidly fragmented by degradation of the ubiquitin-proteasome system, yielding only C-terminal truncated peptides derived from the N-terminal region. Any DRiPs may be used herein.

[0019] In this specification, "polypeptide degradation signal" refers to a tag peptide consisting of a unique amino acid sequence that is linked to the N-terminus or C-terminus of a substrate protein to be degraded, and that induces the protein to enter the ubiquitin-proteasome system. When a ubiquitin ligase recognizes this polypeptide degradation signal and ubiquitinates it, the target substrate protein becomes more easily degraded. Polypeptide degradation signals are not limited to this, but examples include degron sequences. A "degron sequence" is a sequence based on degron technology, in which degradation is induced by a compound (protein degradation inducer). Specific examples of degron sequences include the FKBP12 protein mutant FKBP12 (F36V) and the C-terminal region (cODC) of ornithine decarboxylase.

[0020] The ubiquitin-proteasome system is one of the ATP-dependent protein degradation systems that regulate various biological phenomena in living organisms. In this system, three enzymes—ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligase (E3)—activate ubiquitin, modify target proteins with ubiquitin, and degrade the ubiquitinated target proteins by the proteasome.

[0021] In this specification, "inhibition of protein synthesis" means inhibiting the translation process of mRNA.

[0022] In this specification, "target nucleic acid" refers to a nucleic acid that is the target of the protein synthesis inhibition of the present invention and is involved in mRNA translation in ribosomes. For example, it includes genes and RNA involved in translation. Specifically, although not limited to these, examples include genes encoding aminoacyl-tRNA synthetase (ARS) corresponding to each of the 20 amino acids, genes encoding various ribosomal proteins, tRNA, or rRNA. In this specification, when the target nucleic acid is a gene involved in translation, it will be referred to as the "target gene."

[0023] In this specification, "target polypeptide" refers to a polypeptide that is the target of the protein synthesis inhibition of the present invention and is involved in mRNA translation in ribosomes. Specifically, although not limited to these, examples include aminoacyl-tRNA synthetases (ARS) corresponding to each of the 20 amino acids, and various ribosomal proteins.

[0024] In this specification, "target antigen peptide" means a polypeptide containing a target antigen peptide whose presentation on the cell surface should be enhanced. The target antigen peptide may be a natural protein encoded by a natural gene, or an artificial protein encoded by an artificial gene (such as a chimeric gene) created by gene cloning technology. Furthermore, the target antigen peptide may be a full-length protein or a partial protein.

[0025] In this specification, "expression vector" refers to a vector that contains polynucleotides encoding polypeptides such as proteins in an expressible state and that can control the expression of those polynucleotides. Examples include plasmids. Furthermore, in this specification, "expressible state" refers to placing the target polynucleotide under the control of a promoter within the expression vector. This results in a state where the expression of the target polynucleotide is initiated by the activation of the promoter.

[0026] In this specification, "living organism" means a living cell, tissue, organ, or individual.

[0027] In this specification, "in vitro" means in vitro or ex vivo.

[0028] 1-3. Structure The antigen presentation enhancer of the present invention comprises a protein synthesis inhibitor. The "protein synthesis inhibitor" of the present invention is an agent that has the activity to inhibit the function of genes or proteins involved in mRNA translation in ribosomes, thereby inhibiting peptide chain elongation or reducing translation efficiency.

[0029] Protein synthesis inhibitors, while not limited to these, can consist of, for example, small molecule compounds, nucleic acid molecules, or polypeptides. The following describes the composition of each.

[0030] (1) Small molecule compounds as protein synthesis inhibitors Small molecule compounds used as protein synthesis inhibitors can be any small molecule compounds that target target nucleic acids or target polypeptides involved in mRNA translation in ribosomes and inhibit their function, thereby exhibiting peptide elongation inhibitory activity. Antibiotics are examples of such compounds. Specific examples, though not limited to them, include anisomycin, cycloheximide, puromycin, fusidic acid, and sparsomycin, or their derivatives or salts. Other antiparasitic drugs such as emetine can also be used.

[0031] (2) Nucleic acid molecules as protein synthesis inhibitors Nucleic acid molecules used as protein synthesis inhibitors can be any nucleic acid molecule that targets a target nucleic acid or target polypeptide involved in the mRNA translation process and inhibits its function. Specific examples, though not limited to them, include RNAi agents, antisense oligonucleotides, nucleic acid enzymes, and nucleic acid aptamers. Each of these will be explained below.

[0032] In this specification, "RNAi agent" refers to a nucleic acid substance that induces RNAi in a living organism and suppresses the expression of a target gene (target gene) through the degradation of its transcript or other means. Examples include artificially synthesized dsRNA, siRNA, or shRNA, and endogenous miRNA (microRNA) (including pri-miRNA and pre-miRNA). In this specification, dsRNA or siRNA or shRNA containing a base sequence complementary to a portion of the target gene will be considered the main RNAi agent. Each RNAi agent will be described below.

[0033] "dsRNA" (double-stranded RNA) is a double-stranded RNA consisting of 50 to 1000 nucleotides containing a desired RNA sense strand sequence selected from the sense strand sequence of the target gene. The administered dsRNA is ultimately processed into siRNA (siRNA), as described later, within the cell, causing RNA interference. dsRNA can be designed using known methods based on the nucleotide sequence of the target gene.

[0034] siRNA (small interference RNA) is a small double-stranded RNA molecule consisting of an RNA sense strand (passenger strand) composed of a base sequence corresponding to a portion of the sense strand of a target gene, and an RNA antisense strand (guide strand). siRNA can induce RNA interference by being introduced into the cells (eukaryotic cells) of a test subject. siRNA can be designed using known methods based on the base sequence of the target gene. For example, in siRNA design, a continuous base sequence of 15 to 35 bases, preferably 18 to 25 bases, is selected as the base region for the RNAi sense strand (passenger strand) from the base sequence of the target gene. The base sequence of the RNAi antisense strand (guide strand) should be complementary to the selected RNAi sense strand. When preparing siRNA, the T (thymine) bases in the selected region of both the sense strand and antisense strand are converted to U (uracil) bases. The selected region of the RNAi sense strand is not particularly limited as long as it is a sequence specific to the target gene. Preferably, this is a region at least 50 bases downstream from the start codon, more preferably 70 to 100 bases downstream.

[0035] "shRNA" (short hairpin RNA) is a single-stranded RNA molecule in which two RNA strands (RNAi sense strand and RNAi antisense strand) constituting the siRNA are linked by a spacer sequence consisting of an appropriate base sequence. Within a single molecule, shRNA contains an RNAi sense region as the RNAi sense strand and an RNAi antisense region as the RNAi antisense strand. These regions base-pair to form a stem structure, and the spacer sequence further takes on a loop structure, resulting in a hairpin-shaped stem-loop structure for the entire molecule. When shRNA is introduced into a cell, the loop structure is cleaved to produce a double-stranded RNA molecule, i.e., siRNA. The design of shRNA involves, for example, linking the 3' end of the sense region of the siRNA to the 5' end of the antisense strand with a spacer sequence. The spacer sequence usually consists of 3 to 24 bases, preferably 4 to 15 bases. There are no particular restrictions on the spacer sequence as long as it is a sequence that can base-pair with the siRNA.

[0036] In this specification, "antisense oligonucleotide" refers to a single-stranded nucleic acid molecule composed of a base sequence complementary to all or part of the base sequence of mRNA, which is the transcript of a target gene, and which hybridizes to the target mRNA to suppress its translation. Antisense oligonucleotides are known to exist in both DNA and RNA forms.

[0037] DNA-type antisense oligonucleotides are nucleic acid molecules that hybridize with RNA molecules such as mRNA, which are transcripts of target genes, to form a heteroduplex structure. Subsequently, they suppress the expression of target genes by cleaving and degrading the target RNA molecule through intracellular RNase H activity. DNA-type antisense oligonucleotides are mainly composed of native DNA, but also include native RNA, nucleic acid analogs such as LNA / BNA (Locked Nucleic Acid / Bridged Nucleic Acid) and PMO (Phosphorodiamidate Morpholino Oligomers), and / or modified nucleic acids such as 2'-OMe-RNA, 2'-F-RNA, 2'-MOE-RNA, and phosphorothioates. Furthermore, they also include antisense oligonucleotides with special structures, such as "gapmers," which are RNA-degrading antisense oligonucleotides with wing regions composed of nucleic acid analogs or modified nucleic acids at the 5' and 3' ends, and "mixmers," which are splicing-controlled antisense oligonucleotides composed of nucleic acid analogs or modified nucleic acids.

[0038] Furthermore, heteroduplex nucleic acids utilizing DNA-type antisense oligonucleotides can also be used. A "heteroduplex oligonucleotide" is a double-stranded nucleic acid composed of a main strand (DNA strand) that has antisense oligonucleotide function and a cRNA (complementarily RNA) strand having a base sequence complementary to the main strand. The cRNA strand may also contain nucleic acid analogs or modified nucleic acids, but since at least all or part of the central part of HDO is a heteronucleic acid composed of DNA-RNA, the complementary cRNA strand can be cleaved and degraded by intracellular RNase H, and the remaining main strand can function as an antisense oligonucleotide.

[0039] "RNA-type antisense oligonucleotides" are nucleic acid molecules that hybridize with RNA molecules such as mRNA, which are transcripts of target genes, to form double-stranded RNA. After processing, they ultimately exert a gene expression suppression effect through RNAi, similar to siRNA.

[0040] In this specification, "nucleic acid enzyme" refers to a nucleic acid molecule that possesses catalytic activity. By specifically binding to target mRNA as a substrate and cleaving a specific site, it can suppress the expression of a target gene after transcription and before translation. Known nucleic acid enzymes include deoxyribozymes composed of DNA and ribozymes composed of RNA, and either may be used.

[0041] In this specification, "nucleic acid aptamer" refers to a ligand molecule composed of nucleic acid. It binds strongly and specifically to a target protein through a three-dimensional structure formed based on the secondary and tertiary structures of a single-stranded nucleic acid molecule via hydrogen bonds, thereby inhibiting its function. While RNA aptamers composed of RNA and DNA aptamers composed of DNA are generally known, the nucleic acid aptamer in this specification is not particularly limited and may be either. The base length of the aptamer of the present invention is not limited, but is preferably in the range of 10 to 100 bases. More preferably, it is in the range of 15 to 80 bases.

[0042] Protein synthesis inhibitors may consist of expression vectors. For example, if the protein synthesis inhibitor is an RNA molecule that functions as an shRNA, RNA-type antisense oligonucleotide, ribozyme, or RNA aptamer, an expression vector may be provided that contains the DNA encoding these nucleic acid molecules in a state that allows for expression under promoter control. When such an expression vector is introduced into a target cell, the RNA molecule can be expressed by the activity of the encompassing promoter.

[0043] (3) Polypeptides as protein synthesis inhibitors The polypeptide used as a protein synthesis inhibitor can be any polypeptide that targets a target nucleic acid involved in the translation of mRNA, or a target polypeptide, and inhibits its function. Specific examples, though not limited to them, include antibodies. The antibodies can be natural or artificial. Furthermore, it is preferable that the antibody is a cell-penetrating antibody with cell membrane permeability that allows it to be taken up into cells, but it can also be introduced into cells using commercially available intracellular protein delivery reagents from life science manufacturers. Examples include Chariots (registered trademark: Active Motif).

[0044] "Natural antibodies" refer to antibodies produced in vivo by vertebrates, or antibodies produced from artificially created antibody-producing cells (e.g., hybridoma cells) that have the same amino acid sequence as such antibodies. Natural antibodies include, for example, polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are preferred. "Monoclonal antibodies" refer to a group of clones of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity-determining region, and can recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from hybridomas derived from single cells. The immunoglobulin molecule can be any class (e.g., IgG, IgA, IgE, IgM, IgD, and IgY). Also, like single-chain antibodies produced by camelid animals, which lack a light chain and have a V in the heavy chain... H V, which consists only of regions HH This may include single-chain antibodies, such as antibodies.

[0045] An "artificial antibody" is an antibody that has been artificially constructed. Examples include antibodies in which appropriate mutations have been introduced into the amino acid sequence of the aforementioned natural antibody, as well as single-chain antibodies that have undergone structural modifications that do not exist in nature. A specific example of the latter type of artificial antibody is recombinant antibody. A "recombinant antibody" refers to a chimeric antibody, a humanized antibody, a synthetic antibody, or an antibody fragment.

[0046] A "chimeric antibody" is an antibody created by combining the amino acid sequences of antibodies from different animals, where the variable region (V region) of one antibody is replaced with the V region of another antibody. For example, an antibody in which the V region of a mouse monoclonal antibody is replaced with the V region of a human antibody would be a chimeric antibody, resulting in an antibody with a mouse-derived variable region (V region) and a human-derived C region.

[0047] A "humanized antibody" is a graft antibody in which the complementarity-determining regions (CDRs; CDR1, CDR2, and CDR3) in the variable region (V region) of a suitable mouse antibody, derived from a non-human mammal, are replaced with the CDRs of a human monoclonal antibody. Although the V regions of the heavy and light chains, or the complementarity-determining regions in the V regions of the heavy and light chains of chimeric antibodies and humanized antibodies are derived from non-human animal antibodies such as mice, the C region, or the framework regions (FRs; FR1, FR2, FR3, and FR4) in the V region, and the C regions of the heavy and light chains are derived from human antibodies, thus reducing the immune response to the antibody in the human body.

[0048] A "synthetic antibody" refers to an antibody synthesized by chemical methods or recombinant DNA methods. For example, this includes a monomeric polypeptide molecule or a polymer polypeptide thereof, in which one or more VLs and one or more VHs of a specific antibody are artificially linked via a linker peptide of appropriate length and sequence. Specific examples of such polypeptides include single-chain Fv (scFv: single-chain Fragment of variable region), diabody, triabody, or tetrabody. In immunoglobulin molecules, VL and VH are usually located on separate polypeptide chains (light and heavy chains). A single-chain Fv is a synthetic antibody fragment having a structure in which the V regions on these two polypeptide chains are linked by a sufficiently long flexible linker and incorporated into a single polypeptide chain. Within the single-chain Fv, both V regions can self-assemble to form a single functional antigen-binding site. A single-chain Fv can be obtained by incorporating the recombinant DNA encoding it into a phage genome using known techniques and expressing it. A diabody is a molecule whose structure is based on a single-chain Fv dimer structure. Triabodies and tetrabodies, like diabodies, have trimer and tetramer structures based on a single-chain Fv structure. They are trivalent and tetravalent antibody fragments, respectively, and may be multispecific antibodies. Diabodies and above may be multispecific antibodies. A "multispecific antibody" refers to a multivalent antibody, that is, an antibody that has multiple antigen-binding sites within a single molecule, in which each antigen-binding site binds to a different epitope.

[0049] "Antibody fragments" include, for example, Fab, F(ab')2, and Fv.

[0050] 1-4. Effects In cells treated with the protein synthesis inhibitor, which is an antigen presentation enhancer of the present invention, intracellular protein synthesis is partially inhibited, leading to the production of a large number of DRiPs. In particular, the amount of tDRiPs, which have an N-terminal region that terminates translation early, increases. The increased DRiPs are fragmented by the ubiquitin-proteasome system to form antigen peptides. These antigen peptides can bind to MHC class I molecules and enhance the presentation level expressed on the cell surface.

[0051] Therefore, for example, in cancer immunotherapy utilizing cellular immunity, the antigen presentation enhancer of the present invention can inhibit the peptide elongation reaction of cancer cell-derived proteins and increase the amount of cancer antigen peptides within cells, thereby enhancing the antigen presentation efficiency of cancer antigen peptides to MHC class I molecules expressed on the cell surface. The same applies to viruses. Therefore, the antigen presentation enhancer of the present invention can be a novel immunostimulant that enhances the immune response against cancer or viruses.

[0052] 2. Antigen presentation enhancement composition 2-1. Overview A second aspect of the present invention relates to a composition for enhancing the amount of endogenous peptide-derived antigens presented to MHC class I molecules expressed on the cell surface (often referred to herein as "antigen presentation enhancement composition"). The antigen presentation enhancement composition of the present invention is characterized by containing the antigen presentation enhancement agent described in the first aspect as an active ingredient. The antigen presentation enhancement composition of the present invention makes it possible to maintain the antigen presentation enhancement agent, which is the active ingredient, in an active and stable state and to facilitate its introduction into cells.

[0053] 2-2. Composition The components of the antigen presentation enhancement composition of the present invention will now be described. The antigen presentation enhancement composition of the present invention includes, as an essential component, the antigen presentation enhancement agent described in the first embodiment, which is the active ingredient, and as an optional component, a solvent and / or a carrier. Each component will be described in detail below.

[0054] (1) Active ingredients The antigen presentation enhancement composition of the present invention contains the antigen presentation enhancement agent described in the first embodiment as an active ingredient. The antigen presentation enhancement composition of the present invention may contain one or two or more different types of antigen presentation enhancement agents. Examples include two or more low molecular weight compounds as protein synthesis inhibitors, or a combination of a low molecular weight compound as a protein synthesis inhibitor and a nucleic acid molecule. The specific composition of the antigen presentation enhancement agent is described in detail in the first embodiment, so a detailed explanation is omitted here.

[0055] The amount of active ingredient varies depending on the type of active ingredient, dosage form, and the types of other components described later, so it should be determined appropriately considering each condition. Usually, the dosage is adjusted so that an effective amount of antigen presentation enhancer is included in a single dose administered to cells. Although not limited, for example, if the active ingredient of the antigen presentation enhancer is an antibiotic, it is sufficient that the amount of antigen presentation enhancer present at a concentration of 5% to 40%, 8% to 35%, 10% to 30%, 12% to 25%, or 15% to 20% of the concentration at which protein synthesis is considered to be completely inhibited is included when administered to cells. While not limited, the final concentration should, for example, be in the range of 1 μg / mL to 10 μg / mL, 2 μg / mL to 8 μg / mL, 3 μg / mL to 7 μg / mL, 4 μg / mL to 6 μg / mL, or 4.5 μg / mL to 5 μg / mL for α-amanitin, 0.05 μg / mL to 2.5 μg / mL, 0.08 μg / mL to 2.0 μg / mL, 0.1 μg / mL to 1.5 μg / mL, 0.15 μg / mL to 1.0 μg / mL, 0.2 μg / mL to 0.5 μg / mL, or 0.25 μg / mL to 0.4 μg / mL for anisomycin, or 0.1 mg / mL to 10 mg / mL, 0.4 mg / mL to 8 mg / mL, 0.8 mg / mL to 5 mg / mL, or 1 mg / mL for canavanine. The dosage should be in the range of g / mL to 4 mg / mL, or 2 mg / mL to 2.5 mg / mL; for chloroquine, in the range of 1 μM to 25 μM, 5 μM to 20 μM, 8 μM to 18 μM, 10 μM to 15 μM, or 12.5 μM to 14 μM; for cycloheximide, in the range of 1 μg / mL to 50 μg / mL, 3 μg / mL to 40 μg / mL, 5 μg / mL to 30 μg / mL, 8 μg / mL to 20 μg / mL, or 10 μg / mL to 15 μg / mL; and for MG132, in the range of 0.05 μM to 2.0 μM, 0.08 μM to 1.5 μM, 0.1 μM to 1.0 μM, 0.15 μM to 0.8 μM, or 0.2 μM to 0.5 μM, or 0.25 μM to 0.4 μM.

[0056] (2) Solvent This composition may contain a pharmaceutically acceptable solvent. Examples of solvents include water, aqueous solutions, oils, or organic solvents. Examples of pharmaceutically acceptable aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, sodium acetate buffers, or liquid culture media. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentrations of nonionic surfactants, and polyoxyethylene sorbitan fatty acid esters. Examples of pharmaceutically acceptable oils include natural oils such as vegetable oils. Examples of pharmaceutically acceptable organic solvents include ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.

[0057] (3) Carrier Depending on the dosage form and application, excipients, additives, and / or other active ingredients may be used as pharmaceutically acceptable carriers.

[0058] Excipients include, for example, emulsifiers, pH adjusters, fillers, fluid additive regulators, and lubricants.

[0059] Examples of emulsifiers include surfactants (e.g., polyethylene glycol, poloxamer, glyceryl acetate, isostearamide, sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, propylene glycol fatty acid ester).

[0060] Examples of pH adjusters include alkaline agents such as sodium hydroxide and potassium hydroxide, and acidic agents such as citric acid, sodium citrate, glycolic acid, and ascorbic acid.

[0061] Examples of fillers include petrolatum, the aforementioned sugars, and calcium phosphate.

[0062] Examples of fluid additive regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0063] Other additives include, for example, organic acids, fatty acids, proteins and protein hydrolysates, amino acids and their salts, and vitamins. According to their functional classification, examples include humectants, UV absorbers, antioxidants, stabilizers, preservatives, and bactericides.

[0064] (4) Expression vector The antigen presentation-enhancing composition of the present invention may include an expression vector containing a polynucleotide or antigen protein encoding a target antigen peptide in an expressible state. This target antigen peptide may be any desired polypeptide whose antigen presentation is to be increased on the cell surface to which the antigen presentation-enhancing composition of the present invention is administered. In this case, the target antigen peptide may have a polypeptide degradation signal linked to its N-terminus or C-terminus.

[0065] Examples of target antigen peptides include cancer cell-specific proteins or virus-specific proteins, or peptide fragments of the N-terminal region of these proteins. The amino acid length of the peptide fragment is not limited, but it should be 14-20 amino acids, 12-18 amino acids, 10-15 amino acids, or 8-11 amino acids, including the antigen peptide and a proteasome degradation motif of about 5 amino acids at its C-terminus.

[0066] A "cancer cell-specific protein" refers to a protein that is specifically expressed by cancer cells and can act as a cancer antigen peptide presented on the surface of cancer cells. The type of cancer is not limited as long as it is a cancer cell-specific protein. The type of cancer is also limited to any cancer targeted in cancer immunotherapy. For example, although not limited, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, kidney cancer, tongue cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, testicular cancer, prostate cancer, bladder cancer, brain tumor, leukemia, lymphoma, fibrosarcoma, mast cell tumor, or melanoma may be included. A specific example of the aforementioned cancer cell-specific protein is the MAGE1 protein of melanoma (malignant melanoma).

[0067] A "virus-specific protein" refers to a capsomere protein, which is the unit protein of the capsid that constitutes the outer coat or shell surrounding the viral nucleic acid in a virus particle, or a spike protein contained in the envelope surrounding the capsid. The type of virus is not limited as long as it is a virus-specific protein. The type of virus is also acceptable, as long as it is a virus targeted in immunotherapy. For example, although not limited, viruses that cause viral infections are examples.

[0068] In this specification, "virus" refers to an animal virus that uses animal cells as its host. An animal virus may be either an RNA virus or a DNA virus. For example, if it is an RNA virus, it may be any virus belonging to the Coronaviridae, Orthomyxoviridae, Retroviridae, Picornaviridae, Caliciviridae, Astroviridae, Flaviviridae, Togaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Bunyaviridae, Arenaviridae, Reoviridae, or Birnaviridae families. Specific examples include SARS-CoV-2 of the Coronaviridae family, influenza virus of the Orthomyxoviridae family, or HIV (Human Immunodeficiency Virus) of the Retroviridae family. If it is a DNA virus, it may be any virus belonging to the Papillomaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae, Hepadnaviridae, Circoviridae, Parvoviridae, or Papovaviridae families. A specific example is human papillomavirus of the Papillomaviridae family.

[0069] An antigen presentation-enhancing composition containing an expression vector that encapsulates a polynucleotide encoding the target antigen peptide in an expressible state, when administered to cells, inhibits the translation of the target antigen peptide due to the activity of the active ingredient, the antigen presentation-enhancing agent, and generates a large number of DRiPs of the target antigen peptide. As a result, the amount of the target antigen peptide presented on the surface of the administered cells can be increased.

[0070] 2-3. Dosage Form The dosage form of the antigen presentation enhancing composition of the present invention is not particularly limited as long as it does not inactivate the antigen presentation enhancing agent described in the first embodiment, which is the active ingredient, and other components, and can exert the protein synthesis inhibitory activity of the active ingredient in cells after administration. Furthermore, the specific dosage form of the composition may be appropriately selected depending on the administration method and / or administration conditions. The antigen presentation enhancing composition of the present invention is usually administered to cells or tissues containing them in vitro or ex vivo via cell culture. A liquid formulation is a suitable dosage form for cell administration, although this is not limited.

[0071] 3. Method for producing antigen-enhancing cells 3-1. Overview A third aspect of the present invention is a method for producing antigen-presentation-enhancing cells (often abbreviated as "production method" in this specification) and antigen-presentation-enhancing cells obtained by the production method. The production method of the present invention can produce cells in which the antigen presentation amount of a target antigen peptide is enhanced in vitro.

[0072] 3-2. Method The manufacturing method of the present invention includes an introduction step and an administration step as essential steps, and a culture step as an optional step. Each step will be described in detail below.

[0073] (1)Culture process The "culturing step" is a step in which cells are cultured outside of a living organism. This step can be performed as appropriate before or after the introduction step and administration step described later in the manufacturing method of the present invention.

[0074] The cells provided to the manufacturing method of the present invention are cells that should enhance the amount of antigen presentation of the target antigen peptide on their cell surface. The type of cell is not limited. Examples include primary cultured cells collected from a subject, pluripotent stem cells (iPS cells and ES cells, etc.), adult stem cells (mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, and pigment stem cells, etc.), cell lines, cancer cells, virus-infected cells, etc. The means of obtaining the cells are not limited. For example, they may be collected from a subject or purchased from a life science manufacturer.

[0075] The species from which the cells originate is not limited, but mammals are preferred. Mammals, in this context, include rodents such as mice, rats, hamsters, and guinea pigs; domesticated or pet animals such as dogs, cats, rabbits, cattle, horses, sheep, and goats; and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Humans are particularly preferred.

[0076] This process involves culturing cells using known cell culture media commonly used in cell culture methods in this field. The cell culture media used in this process is not limited, but any highly versatile basic medium used for culturing various types of mammalian cells is acceptable. Specifically, examples include Eagle MEM (Eagle's Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), Ham's Nutrient Mixture F10, Ham's Nutrient Mixture F12, M199 medium, High Performance Medium 199, RPMI-1640 (Roswell Park Memorial Institute-1640), DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F12), and basic neural media. The specific composition of the cell culture medium is publicly known in the field and can be prepared based on the composition described in appropriate literature (e.g., Kaech S. and Banker G., 2006, Nat. Protoc., 1(5):2406-2415). Alternatively, commercially available products manufactured by life science manufacturers such as Thermo Fisher Scientific and Fujifilm Wako Pure Chemical Industries may be used.

[0077] Furthermore, the cell culture medium used in this process may be either serum-containing medium or serum-free medium. In the case of serum-containing medium, the serum may be derived from any species, but is preferably derived from birds or mammals. Examples include human serum and fetal bovine serum (FBS). The preferred concentration of serum is 1-20%, 5-15%, 8-12%, or 10% at the final concentration.

[0078] The cell culture conditions are not particularly limited; cells should be cultured under conditions suitable for the target cells. Specifically, for example, they can be cultured at 37°C under 5% CO2. The culture medium may also be changed every 2 to 4 days as needed.

[0079] (2) Introduction process The "introduction step" is a step in which an expression vector containing a polynucleotide encoding the target antigen peptide in an expressible state is introduced into cells. In this step, transformed cells that express the target antigen peptide by the expression vector are obtained.

[0080] The expression vector containing the polynucleotide encoding the target antigen peptide to be introduced into cells in this process has the same composition as the expression vector described in "2. Antigen Presentation Enhancement Composition; 2-2. Composition: (4) Expression Vector," so its explanation is omitted here.

[0081] The method for introducing the expression vector into cells can be any transformation method known in the field. Examples of transformation methods include lipofection, electroporation, heat shock, PEG, calcium phosphate, DEAE dextran, and protoplast. Alternatively, commercially available intracellular protein delivery reagents from life science manufacturers may be used. For example, Chariots (registered trademark: Active Motif) is one such example.

[0082] (3) Administration process The "administration step" is a step of administering the antigen presentation enhancer described in the first embodiment to the transformed cells obtained in the introduction step. This step is generally performed after the introduction step, but may also be performed simultaneously with the introduction step.

[0083] The specific composition of the antigen presentation enhancer administered in this process is described in detail in the first embodiment, so a detailed explanation is omitted here.

[0084] The antigen presentation enhancer administered in this process may be one type or two or more different types. Alternatively, it may be administered in the form of the antigen presentation enhancer composition described in the second embodiment.

[0085] The administration method is not particularly limited, as long as it allows the cells to be treated with the antigen presentation enhancer. The method should be appropriately considered in relation to the type of antigen presentation enhancer, and administered using methods known in the relevant field. The simplest method of administration, for example, is to add the antigen presentation enhancer to the cell culture medium.

[0086] The dosage of antigen presentation enhancers should be a mild amount that imposes partial translation inhibition on cells. This is because higher dosages strongly inhibit intracellular protein synthesis, resulting in cell death. The specific dosage should be determined appropriately depending on the type of antigen presentation enhancer. For example, if the antigen presentation enhancer is a low-molecular-weight compound such as anisomycin or cycloheximide, as mentioned above, DRiPs can be efficiently produced by adding it at concentrations of 5%-40%, 8%-35%, 10%-30%, 12%-25%, or 15%-20% of the normally used concentration.

[0087] By culturing the transformed cells after this process in the culture step, the target antigen peptide expressed from the expression vector in the cells undergoes inhibition of peptide chain elongation during translation by the antigen presentation enhancer, resulting in the production of a large amount of DRiPs derived from the target antigen peptide within the cells. These DRiPs bind to MHC class I molecules and are efficiently expressed on the cell surface. As a result, cells with enhanced antigen presentation of the target antigen peptide can be produced. [Examples]

[0088] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0089] <Example 1> (the purpose) We will verify the antigen presentation superiority of the N-terminal antigen peptide in vitro.

[0090] (method) Plasmids containing genes encoding antigen proteins fused with the following egg white-derived antigen peptides—pOV8 (SIINFEKL, shown in SEQ ID NO: 1), V-OVA (RGYNYEKL, shown in SEQ ID NO: 2), or R4 (SIIRFEKL, shown in SEQ ID NO: 3)—were introduced into mouse-derived cultured cells EL4-Luc2 obtained from ATTC, and these were expressed. For the fused antigen proteins, the amino acid sequences shown in SEQ ID NO: 4 (LEQLE) and 5 (TEWTS), respectively, were added as 5 flanking amino acids to the N-terminus and C-terminus of the egg white-derived antigen peptides pOV8, V-OVA, and R4, respectively, before fusing them with the fluorescent proteins.

[0091] The expression level of antigen proteins in each cell was quantified by fluorescence intensity, and the formation of the MHC class I molecule-pOV8 complex presented on the cell surface MHC class I molecule was identified, along with the formation of the complex (pOV8 / H-2K b Anti-H-2K specific to the complex b Using the (SIINFEKL) antibody (25-D1.16; donated by Dr. Germain: Porgador, A., et al., 1997, Immunity, 6, 715-726), FACS (BD FACS Canto) was performed. TM The mean fluorescent intensity (MFI) of II was used for quantification, and the correlation between the expression level of the antigen protein and the presentation of the antigen peptide derived from that antigen protein was compared. The same instrument and quantification method were used for all subsequent FACS studies.

[0092] (result) The results are shown in Figures 1-1 and 1-2. Each figure shows the relative values ​​with the MFI value set to 1 in negative control cells into which a plasmid containing a gene encoding only the fluorescent protein (YFP or AZ) was introduced.

[0093] Figure 1-1 is a schematic diagram of the fluorescent protein fusion antigen protein constructed and used in this example. Figures 1-2a to e show the expression level of the antigen protein and the presentation level of the antigen peptide derived from that antigen protein in this example.

[0094] From Figure 1-2a, when pOV8 is located at the N-terminus of YFP, compared to the opposite case, it is found in MHC class I molecules (H-2K b It was shown that the antigen is more readily presented on the molecule (p<0.01). This difference was also verified in cell lines other than EL4-Luc2 cells (DC2.4 and MC57G), and the results were similar (data not shown). Furthermore, no antigen presentation was observed in negative controls expressing only YFP. In addition, although not shown in the figures, when only pOV8 was expressed, no fluorescence was detected, but antigen presentation was observed.

[0095] Figure 1-2b shows the results of an investigation into the case where the egg white-derived antigen peptide pOV8 from a above was replaced with V-OVA and fused to YFP. The same trend as in a was observed even when the egg white-derived antigen peptide was changed (p<0.01).

[0096] Figure 1-2c shows the results of further testing when the egg white-derived antigen peptide pOV8 from a above was fused to YFP instead of R4. As a result, a similar trend to a was confirmed (p<0.01).

[0097] Figure 1-2d shows the results of an investigation into the case where the fluorescent protein YFP in a was replaced with AZ and fused to pOV8. The results showed a similar trend to a (p<0.01).

[0098] Figure 1-2e shows the results of testing various fusion patterns of the fusion protein containing two sets of the fluorescent protein YFP (a). The results showed that the pOV8-YFP-YFP fusion protein, with pOV8 at the N-terminus, had the highest antigen presentation, followed by the YFP-pOV8-YFP fusion protein, with pOV8 in the center, and the YFP-YFP-pOV8 fusion protein, with pOV8 at the C-terminus, had the lowest antigen presentation (p<0.01).

[0099] From these results, it became clear that antigen presentation efficiency is significantly higher in the N-terminal region of the antigen protein than in the C-terminal region, regardless of the type of antigen protein. In other words, this suggests that the amount of antigen peptide presented is inversely proportional to the distance from the N-terminus of the antigen protein.

[0100] <Example 2> (the purpose) The superiority of the N-terminal antigen peptide will be verified both ex vivo and in vivo.

[0101] (method) The experiment involved cytotoxic T cells (MagniSort) purified one week after immunization from OT-I mice immunized with pOV8 (transgenic mice expressing a TCR that recognizes MHC class I molecules presenting pOV8). TM The Mouse CD8 T cell Enrichment Kit (Thermo Fisher Scientific) was co-cultured for 24 hours with EL4-Luc2 cells stably expressing YFP, pOV8-YFP, or YFP-pOV8, respectively, in various ratios. The EL4-Luc2 cell to cytotoxic T cell ratio (ET ratio) was set to 0:1, 1:1, 5:1, 10:1, 50:1, and 100:1. After 24 hours of co-culture, the number of cells that underwent cell death was specifically stained with 7-AAD, and the number of cells that underwent antigen-specific cell death was measured using FACS to determine the percentage of cells that underwent antigen-specific apoptosis.

[0102] Next, OT-I mice were transplanted with equal numbers of EL4-Luc2 cells stably expressing YFP, pOV8-YFP, or YFP-pOV8, and then immunized with pOV8. On the day of immunization (Day 0) and two weeks later (Day 14), the distribution of EL4-Luc2 cells within the mouse was detected using IVIS Lumina (Sumisho Pharma International) by the luminescence of luciferase expressed by EL4-Luc2 cells, and this was quantified to confirm the in vivo proliferation of each type of EL4-Luc2 cell.

[0103] Furthermore, EL4-Luc2 cells expressing three different antigenic peptides were transplanted into OT-I mice, and the survival rate of OT-I mice immunized with pOV8 was calculated.

[0104] (result) The results are shown in Figures 2-1 to 2-3. Figure 2-1 shows the percentage of cells that underwent antigen-specific cell death when EL4-Luc2 cells stably expressing three types of antigen proteins were co-cultured with cytotoxic T cells at their respective ET ratios.

[0105] When the ET ratio was 0:1, 1:1, and 5:1, no significant difference was observed in the proportion of apoptotic cells between pOV8-YFP-expressing cells and YFP-pOV8-expressing cells. However, when the ET ratio was 10:1 or higher (10:1, 50:1, and 100:1), pOV8-YFP-expressing cells underwent antigen-specific apoptosis at a significantly higher rate compared to YFP-pOV8-expressing cells (p<0.01). This result indicates that the superiority of the N-terminal antigen peptide is effective in inducing apoptosis of target cells by cytotoxic T cells, even in exovivotry.

[0106] After transplanting equal numbers of each of the three types of EL4-Luc2 cells into OT-I mice, the distribution and number of EL4-Luc2 cells within the mouse body from the day of pOV8 immunization (Day 0) to two weeks later (Day 14) showed no significant differences on Day 0 (not shown). However, on Day 14, EL4-Luc2 cells expressing only YFP without presenting pOV8 increased in number and spread widely throughout the body (not shown). On the other hand, EL4-Luc2 cells expressing pOV8-YFP (presenting pOV8) and YFP-pOV8, respectively, decreased in number on Day 14 compared to Day 0, indicating that the distribution of EL4-Luc2 cells within the body was extremely limited (not shown). Furthermore, the luminescence of EL4-Luc2 cells expressing pOV8-YFP was lower than that of EL4-Luc2 cells expressing YFP-pOV8.

[0107] Figure 2-2 shows the results of quantifying the luminescence of luciferase expressed by EL4-Luc2 cells in mice, and verifying the in vivo proliferation of EL4-Luc2 cells expressing three different antigen proteins. On Day 0, no significant difference was observed in the luminescence of YFP-expressing cells, pOV8-YFP-expressing cells, and YFP-pOV8-expressing cells. On Day 14, however, YFP-expressing cells proliferated, while YFP-pOV8-expressing cells significantly decreased (p<0.01). pOV8-YFP-expressing cells were even more significantly fewer than YFP-pOV8-expressing cells (p<0.01). These results indicate that the superiority of the N-terminal antigen peptide was effective in the immune response against target cells by cytotoxic T cells, even in vivo.

[0108] Figure 2-3 shows the survival rates of OT-I mice that were transplanted with equal numbers of EL4-Luc2 cells expressing the three types of antigen peptides, respectively, and then immunized with pOV8. From this figure, it was clear that OT-I mice transplanted with pOV8-YFP-expressing cells survived for the longest period.

[0109] These results all suggest that the superiority of the N-terminal antigen peptide is established not only in vitro but also in ex vivo and in vivo, indicating that the superiority of the N-terminal antigen peptide plays a role in the immune response.

[0110] <Example 3> (the purpose) We will investigate the amount of antigen presented when mature proteins are transfected into cells. Assuming that the superiority of the N-terminal antigen peptide originates from DRiPs, it is predicted that this superiority will be lost when purified mature proteins are directly introduced into cells. Therefore, in this example, we will investigate the amount of antigen presented when mature proteins expressed in E. coli are directly transfected into cells.

[0111] (method) Expression vectors encoding the YFP, pOV8-YFP, and YFP-pOV8 antigen proteins were expressed in E. coli, purified with His-tag, and endotoxin was removed using NEA-1000R (WAKO). Subsequently, each antigen protein was introduced into EL4-Luc2 cells using Chariots (registered trademark: Active Motif; hereafter the same) according to the attached protocol, and anti-H2K b The amount of antigen presented was quantified by FACS using the (SIINFEKL) antibody (25-D1.16: as described above).

[0112] Furthermore, mature proteins introduced into EL4-Luc2 cells were detected by YEP-based Western blotting, and their intracellular protein levels were quantified.

[0113] (result) The results are shown in Figures 3-1 and 3-2. Figure 3-1 shows the relative amounts of transfected proteins (YFP, pOV8-YFP, and YFP-pOV8) in EL4-Luc2 cells, with the amount of YFP (No. 5) introduced using Chariots set to 1. In the figure, Chariots (registered trademark: Active Motif) is a protein transfection reagent, and MG132 is a proteasome inhibitor.

[0114] When three types of mature proteins were added in the absence of Chariots (No. 2-4), no significant difference in protein quantity was observed compared to the control group (No. 1) without mature proteins. On the other hand, when pOV8-YFP (No. 6) or YFP-pOV8 (No. 7) were introduced in the presence of Chariots, antigen presentation was confirmed (p<0.01). This antigen presentation disappeared upon the addition of the proteasome inhibitor MG132 (No. 9-10). Therefore, it was suggested that the mature proteins introduced into the cells were presented on the cell surface via the DP (direct presentation) pathway. Furthermore, as predicted, no significant difference was observed between pOV8-YFP (No. 6) and YFP-pOV8 (No. 7), revealing that the superiority of the N-terminal antigen peptide is lost.

[0115] Figure 3-2 shows the amount of three antigen proteins, YFP, pOV8-YFP, and YFP-pOV8, taken up by EL4-Luc2 cells, detected and quantified by Western blotting using an antibody against YFP. The concentration of each protein band obtained by Western blotting was corrected for the concentration of the β-actin band for quantification. No significant difference was observed in the amounts of pOV8-YFP and YFP-pOV8 taken up by EL4-Luc2 cells. Furthermore, the intracellular amounts of the three antigen proteins did not change significantly even with the addition of MG132.

[0116] These results strongly suggest that the superiority of N-terminal antigenic peptides does not result from the difference in intracellular stability of the mature protein.

[0117] <Example 4> (Objective) The effects of various inhibitors on the expression of H2-Kb / pOV8 were investigated. The effects of various inhibitors on the antigen presentation of N-terminal antigenic peptides are verified.

[0118] (Method) EL4-Luc2 cells stably expressing YFP, pOV8-YFP, and YFP-pOV8 respectively were added with various inhibitors (α-amanitin, anisomycin, canavanine, chloroquine, cycloheximide, emetine, MG132) and cultured, and 12 hours later, the expression level of H-2K b / pOV8 was quantified using the 25-D1.16 antibody. Note that α-amanitin (αAm.) is an RNA synthesis inhibitor that stops transcription. Anisomycin (Ani.), cycloheximide (CHX), or emetine (Eme.) are all ribosome inhibitors that inhibit protein translation at the ribosome. Canavanine (Cana.) is an analog of arginine. Furthermore, chloroquine (Chlo.) is a lysosomal protease inhibitor, and MG132 is a proteasome inhibitor.

[0119] (Result) The results are shown in Figure 4-1 and Figure 4-2. Figure 4-1 is a diagram showing the amount of antigen presentation on the cell surface in the presence of various inhibitors. In addition, Figure 4-2 is a diagram showing the ratio of the antigen presentation amounts of pOV8-YFP and YFP-pOV8 on the cell surface in the presence of various inhibitors.

[0120] As shown in Figure 4-1, when transcription was stopped by inhibiting RNA synthesis using α-amanitin (αAm.), the amount of pOV8 presented decreased in both pOV8-YFP-expressing cells and YFP-pOV8-expressing cells. However, as shown in Figure 4-2, the ratio of pOV8 presented in pOV8-YFP-expressing cells to YFP-pOV8-expressing cells did not show a significant difference compared to the control experiment. This result suggests that pOV8 presented on MHC class I molecules depends on new RNA synthesis and protein synthesis. It also strongly suggests that the pOV8 peptide is not supplied by the degradation of mature pOV8-YFP or YFP-pOV8 proteins already present in the intracellular cytosol.

[0121] In contrast, inhibition of protein translation in ribosomes using anisomycin (Ani.), cycloheximide (CHX), or emetine (Eme.) significantly increased the amount of pOV8 presented in pOV8-YFP-expressing cells compared to the control experiment, as shown in Figure 4-1, but no significant difference was observed in YFP-pOV8-expressing cells. Furthermore, as shown in Figure 4-2, the ratio of pOV8 presentation in pOV8-YFP-expressing cells to that of YFP-pOV8-expressing cells was significantly increased compared to the control experiment (p<0.001). This result suggests that the aforementioned translation inhibitors inhibited protein translation in ribosomes and interrupted peptide chain elongation, leading to an increase in tDRiPs. Consequently, while antigen presentation from pOV8-YFP was enhanced, antigen presentation from YFP-pOV8 had little effect, or rather, it was inhibited by reducing the amount of mDRiPs.

[0122] Conversely to the ribosome inhibitor, when canavanine (Cana.), an analog of arginine, was added, the amount of pOV8 presented by both pOV8-YFP-expressing cells and YFP-pOV8-expressing cells significantly increased, as shown in Figure 4-1. However, as shown in Figure 4-2, there was no significant difference in the ratio of pOV8 presentation between pOV8-YFP-expressing cells and YFP-pOV8-expressing cells compared to the control experiment. Canavanine is mistakenly incorporated into proteins as arginine, but proteins that incorporate canavanine cannot assume a normal three-dimensional structure, resulting in an increase in both tDRiPS and mDRiPs. As a result, while the amount of pOV8 antigen presented increased in both pOV8-YFP and YFP-pOV8, the ratio of pOV8 presentation between pOV8-YFP and YFP-pOV8 did not show a significant difference compared to the control experiment.

[0123] Finally, as shown in Figure 4-1, chloroquine (Chlo.), a lysosomal protease inhibitor, significantly increased the amount of pOV8 presented by both pOV8-YFP-expressing cells and YFP-pOV8-expressing cells. On the other hand, as shown in Figure 4-2, the ratio of pOV8 presentation between pOV8-YFP and YFP-pOV8 did not show a significant difference compared to the control experiment. DRiPs and mature proteins are also degraded by autophagy, but autophagy-mediated degradation of antigen proteins does not usually contribute to antigen presentation. However, the above results suggest that inhibition of autophagy function increased the amount of antigen protein degraded by the proteasome and supplied for antigen presentation. In contrast, as shown in Figure 4-2, MG132 reduced the presentation of pOV8 in both the pOV8-YFP and YFP-pOV8 ratios, but as shown in Figure 4-2, the ratios between pOV8-YFP and YFP-pOV8 did not show a significant difference compared to the control experiment.

[0124] These results strongly suggest that pOV8 presented on MHC class I molecules is dependent on the classical DP pathway.

[0125] <Example 5> (the purpose) The results of the inhibitor experiment in Example 4 suggested that the increase in tDRiPs due to ribosome translation inhibition enhances the dominance of the N-terminal antigen peptide. Therefore, we will investigate the effect on this dominance from a molecular biological perspective.

[0126] (method) Inhibition of molecules that constitute ribosomes strongly affects cell survival, and under severe conditions, it can induce cell death. Therefore, to investigate the effect on antigen presentation, we partially inhibited the translation process in ribosomes by suppressing the gene expression of aminoacyl-tRNA synthetase, which interferes with the formation of a covalent bond between the 3' end of tRNA and an amino acid, using RNAi. In this example, the expression of alanyl-tRNA synthetase (AlaRS), one of the aminoacyl-tRNA synthetases, was specifically reduced using AlaRS siRNA(m) (sc-72475; Santa Cruz Biotechnology). The amount of antigen presented on each cell surface was quantified by FACS using a 25-D1.16 antibody.

[0127] (result) The results are shown in Figures 5-1 and 5-2. Figure 5-1 shows the relationship between RNAi-mediated inhibition of alanyl-tRNA synthetase translation and antigen presentation. Figure 5-2 compares the changes in antigen presentation of pOV8-YFP and YFP-pOV8 due to RNAi-mediated inhibition of alanyl-tRNA synthetase expression. As shown in Figure 5-1, the amount of pOV8 presented by pOV8-YFP-expressing cells increased significantly, but conversely, as shown in Figure 5-2, there was no significant difference in the amount of pOV8 presented by YFP-pOV8-expressing cells compared to the control experiment (p<0.001). As a result, the ratio of pOV8 presentation between pOV8-YFP-expressing cells and YFP-pOV8-expressing cells was significantly wider than in the control experiment (p<0.001).

[0128] Western blotting using specific antibodies confirmed that alanyl-tRNA synthetase expression was suppressed and reduced by RNAi, and that this resulted in a slight decrease in the expression of YFP, pOV8-YFP, and YFP-pOV8, although there was no significant difference between the expression levels of pOV8-YFP and YFP-pOV8 (data not shown).

[0129] <Example 6> (the purpose) We will investigate how the enhancement of N-terminal antigen peptide dominance through translation inhibition functions in cellular immunity.

[0130] (method) OT-I mice immunized with pOV8 (pOV8-H2K b Cytotoxic T cells (MagniSort) purified one week after immunization from transgenic mice expressing a TCR that recognizes the complex. TM Mouse CD8 T cell enrichment kits (Thermo Fisher Scientific) were pre-cultured for 12 hours in media containing the translation inhibitors anisomycin, cycloheximide, and emetine, respectively. Subsequently, these cells were co-cultured for 24 hours with EL4-Luc2 cells stably expressing YFP, pOV8-YFP, or YFP-pOV8 at an ET ratio of 5:1, and the number of cells that underwent cell death was specifically stained with 7-AAD. The percentage of antigen-specific apoptotic cells was then measured by FACS.

[0131] Next, following the same procedure as in Example 2, equal numbers of EL4-Luc2 cells stably expressing YFP, pOV8-YFP, or YFP-pOV8 were transplanted into OT-I mice, and then immunized with pOV8. Two weeks after immunization, the distribution of EL4-Luc2 cells within the mice was detected using IVIS Lumina (Sumisho Pharma International) by detecting the luminescence of luciferase expressed by EL4-Luc2 cells, and this was quantified to confirm the proliferation of each EL4-Luc2 cell type in vivo.

[0132] (result) The results are shown in Figures 6-1 and 6-2. As shown in Figure 6-1, in the control experiment without the addition of a translation inhibitor, there was no significant difference in the proportion of apoptotic cells between OV8-YFP-expressing cells and YFP-pOV8-expressing cells. In contrast, the proportion of apoptotic cells increased in pOV8-YFP-expressing cells pre-cultured in the presence of a translation inhibitor. On the other hand, in YFP-pOV8-expressing cells, no significant change in this proportion was observed compared to the control group. From these results, it was revealed that the addition of a translation inhibitor induces significantly higher antigen-specific apoptosis in OV8-YFP-expressing EL4-Luc2 cells (p<0.001).

[0133] Figure 6-2 shows that pOV8-YFP-expressing cells without the addition of translation inhibitors proliferated, while pOV8-YFP-expressing cells with the addition of translation inhibitors (anisomycin, emetine, and cycloheximide) significantly decreased. This result suggests that the addition of translation inhibitors enhances the dominance of the N-terminal antigen peptide, leading to more efficient induction of apoptosis of target cells by cytotoxic T cells.

[0134] From the above results, it became clear that the antigen presentation efficiency is significantly enhanced by the translation inhibition of the present invention in the region located at the N-terminus of the antigen protein, while no significant enhancement in antigen presentation efficiency was observed in the region located at the C-terminus of the antigen protein. Therefore, for example, it became clear that a method of enhancing the presentation amount of cancer antigen peptides located at the N-terminus by translation inhibition of the present invention is effective in enhancing the immune response against cancer cells.

Claims

1. A protein synthesis inhibitor that enhances the presentation of endogenous peptide-derived antigens to MHC class I molecules expressed on the cell surface.

2. The antigen presentation enhancer according to claim 1, wherein the endogenous peptide is derived from DRiPs (defective ribosomal products).

3. The antigen presentation enhancer according to claim 2, wherein the DRiPs are tDRiPs.

4. The antigen presentation enhancer according to any one of claims 1 to 3, wherein the protein synthesis inhibitor comprises a low molecular weight compound, a nucleic acid, or a polypeptide.

5. The antigen presentation enhancer according to claim 4, wherein the low molecular weight compound is a substance having peptide elongation inhibitory activity in ribosomes.

6. The antigen presentation enhancer according to any one of claims 1 to 5, wherein the cells are cancer cells or virus-infected cells.

7. A composition for enhancing the amount of antigen presented to MHC class I molecules in cells, comprising an antigen presentation enhancer according to any one of claims 1 to 6, derived from endogenous peptides.

8. The antigen presentation enhancement composition according to claim 7, comprising an expression vector containing a polynucleotide encoding a target antigen peptide in an expressible state.

9. The antigen presentation enhancement composition according to claim 8, wherein the target antigen peptide is a cancer cell-specific protein or a virus-specific protein, or a peptide fragment of its N-terminal region.

10. The antigen presentation enhancement composition according to claim 8 or 9, wherein a polypeptide degradation signal is linked to the terminus of the target antigen peptide.

11. A cellular immune-stimulating composition comprising the antigen presentation enhancement composition described in any one of claims 7 to 10 as an active ingredient.

12. A method for producing cells in which the antigen presentation amount of a target antigen peptide is enhanced in vitro, An introduction step of introducing an expression vector containing a polynucleotide encoding a target antigen peptide in an expressible state into the cells, and Administration step: Administering the transformed cells obtained in the above introduction step to the antigen presentation enhancer according to any one of claims 1 to 6. The method comprising the above.

13. The manufacturing method according to claim 12, wherein the cells are cells collected from a human.

14. The method for producing the target antigen peptide according to claim 12 or 13, wherein the target antigen peptide is a cancer cell-specific protein or a virus-specific protein, or a peptide fragment of its N-terminal region.

15. Antigen presentation-enhancing cells obtained by the manufacturing method described in any one of claims 12 to 14.

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