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WO2024262558A1 - Peptide d'antigène tumoral codé par un gène de rétrovirus endogène humain - Google Patents

Peptide d'antigène tumoral codé par un gène de rétrovirus endogène humain Download PDF

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WO2024262558A1
WO2024262558A1 PCT/JP2024/022321 JP2024022321W WO2024262558A1 WO 2024262558 A1 WO2024262558 A1 WO 2024262558A1 JP 2024022321 W JP2024022321 W JP 2024022321W WO 2024262558 A1 WO2024262558 A1 WO 2024262558A1
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cancer
tumor
peptide
antigen
present
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貴幸 金関
俊彦 鳥越
芹奈 時田
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Sapporo Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a detection agent for detecting tumor cells that utilizes a gene that is specifically expressed in tumor cells, a tumor antigen peptide derived from the gene that is useful as a cancer prevention and/or treatment agent, and uses thereof.
  • CD8 + T cells can recognize tumor antigens presented by HLA class I molecules and eliminate tumor cells.
  • Immune checkpoint blockade is often beneficial in renal cell carcinoma (RCC) patients, regardless of their low tumor mutation burden (TMB).
  • CD8 + T cells can distinguish tumor cells from normal cells and eliminate them. CD8 + T cells often recognize HLA-presented neoantigens that arise from somatic gene mutations. Neoantigens are truly specific to tumor cells, and neoantigen-reactive T cells are not eliminated by negative selection in the thymus. As a result, neoantigens can induce host antitumor responses in various tumors and serve as targets for activated T cells after ICB (Non-Patent Documents 1-3). Thus, the mutation load in tumors positively correlates with patient survival after ICB (Non-Patent Document 4). Similarly, tumors with histological types that have a high mutation load, such as melanoma and non-small cell lung cancer, are sensitive to ICB (Non-Patent Document 5).
  • Non-Patent Document 6 Merkel cell carcinoma and renal cell carcinoma are exceptions to this trend. ICB responses to these tumors were better than predicted based on TMB. Because the development of Merkel cell carcinoma is associated with viral infection, ICB responses may result from T cell recognition of Merkel cell polyomavirus (Non-Patent Document 7). In contrast, the mechanism by which T cells recognize RCC is elusive.
  • hERVs human endogenous retroviruses
  • Non-Patent Document 8 retroviral germline gene-encoded elements that account for approximately 8% of the human genome
  • Non-Patent Document 9 hERVs are inactive or dysfunctional under physiological conditions, but some retain protein-coding potential.
  • reactivated hERVs appear to elicit cytotoxic T cell responses in RCC (Non-Patent Document 10).
  • Non-Patent Document 11 This idea is supported by the positive correlation between hERV expression and clinical response to ICB in RCC patients (Non-Patent Document 11), which may contribute to the activation of both innate and adaptive immunity, providing evidence for hERV-derived antigens and T cell recognition (Non-Patent Document 12).
  • Such findings strongly suggest a role for hERV in T cell-mediated antitumor responses in RCC.
  • T cell recognition of hERV-derived antigens was reported in melanoma patients in 2002 (Non-Patent Document 13), the technical difficulties associated with defining hERV-derived antigens have prevented comprehensive analysis of this unique class of peptides.
  • Patent Document 1 it was found that a partial peptide of a polypeptide predicted to be encoded by the open reading frame (ORF) of PVT1 is presented as an antigen in cancer cells, and that HF10 derived from the ORF of PVT1 is useful for immunotherapy such as cancer vaccines.
  • ORF open reading frame
  • Patent Document 1 also describes that a partial peptide of a protein encoded by a gene selected from the group consisting of SUV39H2, ZNF724P, SNRNP40, and DYRK4, or the PVT1 peptide, is presented by HLA, and describes a CTL inducer containing the partial peptide as an active ingredient, but does not describe the use of proteogenomic HLA ligandome analysis together with mass spectrometry (MS) and next-generation sequencing to explore the immune peptidome of tumor tissue and identify hERV-derived antigens.
  • MS mass spectrometry
  • Alexandrov LB Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, B orresen-Dale AL, et al. Signatures of mutational processes in human cancer. Nature. 2013;500(7463):415-21. Yarchoan M, Hopkins A, and Jaffee EM. Tumor Mutational Burden and Response Rate to PD-1 Inhibition. N Engl J Med. 2017;377(25):2500-1.
  • Endogenous retrov irus expression is associated with response to immune checkpoint blockade in clear cell renal cell carcinoma. JCI Insight. 2018;3(16). Smith CC, Beckermann KE, Bortone DS, De 1 Cubas AA, Bixby LM, Lee SJ, Panda A, Ganesan S, Bhanot G, Wallen EM, et al. Endogenou s retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma. J Clin Invest. 2018;128(11):4804-20. Schiavetti F, Thonnard J, Colau D, Boon T, and Coulie PG. A human endogenous retroviral sequence encoding an antigen recognized on melanoma by cytolytic T lymphocytes. Cancer Res. 2002;62(19):5510-6.
  • the object of the present invention is to provide a detection agent for specifically detecting tumor cells, a tumor antigen peptide that is specifically presented by tumor cells, and a pharmaceutical composition containing the same as an active ingredient and useful for preventing and/or treating cancer.
  • the inventors also compared renal tumor and normal tissues and identified tumor-associated hERV antigens, one of which was found to be immunogenic and recognized by host tumor-infiltrating lymphocytes (TILs).
  • TILs host tumor-infiltrating lymphocytes
  • PBMCs peripheral blood mononuclear cells
  • HD healthy donors
  • the present invention relates to the following: [1] A tumor antigen peptide or a motif substitution thereof, which consists of 8 to 14 consecutive amino acids in the amino acid sequence of a protein encoded by a human endogenous retrovirus (hERV) gene and has HLA-binding activity. [2] The tumor antigen peptide or its motif substitution of [1], wherein the HLA is HLA-A24. [3] The tumor antigen peptide of [1] or [2] or a motif substitution thereof, wherein the protein encoded by the human endogenous retrovirus (hERV) gene is represented by SEQ ID NO: 2.
  • [6] A polyepitope peptide in which multiple epitope peptides are linked, the polyepitope peptide comprising at least one of the tumor antigen peptides according to [1] to [5].
  • [7] A polynucleotide encoding at least one of the tumor antigen peptides of [1] to [5], the polyepitope peptide of [6], or the protein represented by SEQ ID NO: 2 or a fragment thereof containing LYDTVTHTF (SEQ ID NO: 3).
  • A a tumor antigen peptide according to any one of [1] to [5] or a polyepitope peptide according to [6];
  • B a polynucleotide encoding at least one of the peptides and/or polyepitope peptides of (A), or
  • C A method for inducing cytotoxic T cells, comprising contacting in vitro antigen-presenting cells that present any one of the tumor antigen peptides [1] to [5] as an antigen with peripheral blood lymphocytes.
  • a diagnostic agent comprising the HLA multimer of [19].
  • a T cell receptor-like antibody that recognizes a complex of any one of the antigen peptides [1] to [5] and HLA.
  • a tumor detection agent comprising the T cell receptor-like antibody of [21].
  • a chimeric antigen receptor that recognizes a complex of any one of the antigen peptides [1] to [5] and HLA.
  • An artificial CTL comprising a T cell receptor that recognizes a complex of any one of the antigen peptides [1] to [5] and HLA.
  • a bispecific antibody that specifically recognizes a complex of an antigen peptide of any one of [1] to [5] and HLA and a lymphocyte surface antigen.
  • a tumor cell detection agent comprising a detection agent for detecting an expression product of a human endogenous retrovirus (hERV) gene.
  • a tumor cell detection agent for detecting tumor cells from one or more tumor samples selected from the group consisting of gastrointestinal cancer, breast cancer, uterine cancer, soft tissue tumors, bladder cancer, head and neck cancer, skin cancer, lung cancer, colon cancer, brain cancer, heart cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer, spleen cancer, thymus cancer, prostate cancer, ovarian cancer, and blood cancer.
  • the expression product of the gene is mRNA, and the agent comprises a probe and/or a primer having a base sequence complementary to the gene.
  • the tumor cell detection agent according to any one of [26] to [28], wherein the expression product of the gene is an endogenous polypeptide, and the agent contains a detection substance that specifically reacts with the endogenous polypeptide.
  • An siRNA comprising an antisense region complementary to a human endogenous retrovirus (hERV) gene and a sense region at least partially complementary to the antisense region.
  • a pharmaceutical composition comprising the antisense oligonucleotide of [34] and/or the siRNA of [35], and a pharma- ceutically acceptable carrier.
  • the pharmaceutical composition of [36] which is a preventive and/or therapeutic agent for cancer.
  • the present invention highlights the existence of antitumor CD8 + T cell surveillance against hERV antigens and suggests their clinical application in renal cell carcinoma (RCC) patients.
  • RRC renal cell carcinoma
  • the present invention provides a tumor antigen peptide useful as an inducer of CTL that specifically attacks tumor cells, and a pharmaceutical composition containing the same as an active ingredient, which is useful for preventing and/or treating cancer, and the like.
  • FIG. 1A shows the expression of immune-related genes in RCC tissues (RCC17, RCC19, and RCC21).
  • Figure 1B shows a section of an RCC17 kidney showing the tumor (T) and normal (N) lesions used in this study. The dotted line indicates the capsule surrounding the tumor mass.
  • FIG. 1C shows a comparison of the expression levels of genes associated with cytotoxic T cells (top panel) and costimulatory or immune checkpoint molecules (bottom panel) between normal and tumor tissues of RCC17.
  • FIG. 1D shows immunohistochemistry of RCC17 tumor and normal tissues (magnification ⁇ 200).
  • Figure 1E shows the number of CD8 + T cells per HPF in RCC17, RCC19, and RCC21 tissues. Box plots represent median (solid line), quartiles (boxes), and 1.5x quartiles (vertical lines). p values were calculated using two-tailed t-tests. ( *** p ⁇ 0.001, ** p ⁇ 0.01)
  • FIG. 2A shows the workflow of the proteogenomic analysis exploring normal and tumor tissues.
  • Peptide-HLA-A24 complexes were captured from RCC17 tumor and normal tissues using specific antibodies, and the eluted peptides were subsequently analyzed by MS.
  • a personalized database was used to allow detection of peptides derived from hERV by MS sequencing.
  • Figure 2B shows the distribution of lengths of reference peptides identified in normal and tumor tissues, with each bar showing the number of peptides identified.
  • FIG. 2C shows the logo sequence indicating the conserved amino acid at each position across all 9-mer peptides.
  • Figure 2D shows a violin diagram showing the %rank score (NetMHCpan4.1) of identified peptides.
  • FIG. 2E shows the length distribution of hERV-derived peptides identified in normal and tumor tissues, with each bar showing the number of peptides identified.
  • FIG. 2F shows a pie chart depicting the frequency of hERV-derived peptides among the identified peptides.
  • Figure 2G shows a violin diagram showing the nucleotide lengths of the source ORFs encoding the identified peptides. The round dots indicate the distribution of ORFs encoding hERV-derived peptides.
  • the box plots represent the median (solid line), quartiles (boxes), and 1.5x quartiles (vertical lines). Dots indicate observations outside the range of adjacent values.
  • FIG. 3A shows a Venn diagram showing the number of hERV-derived peptides exclusive to RCC17 tumor and normal tissues.
  • Figure 3B shows differential gene expression of hERV in tumor and normal tissues. Tumor-exclusive hERV-encoded peptides are indicated by dots.
  • Figure 3C shows gene expression of four hERV transcripts across three RCC tissues. Each fold change (FC) represents the expression ratio (tumor/normal) in RCC17.
  • FIG. 3D shows hERV3895 expression across normal tissues as measured by RT-PCR (Y-axis is FC relative to RCC21 normal tissue).
  • Figure 3E shows a schematic of the herv3895 transcript. Each black box indicates a potential ORF encompassed by an ATG and a stop codon.
  • FIG. 3F shows the MS/MS spectra and corresponding b and y fragment ions of the endogenous and synthetic LF9 peptides.
  • FIG. 4A shows flow cytometry of tumor infiltrating T lymphocytes (TILs) obtained from RCC17 tumor tissue. TILs were analyzed after in vitro expansion. Data are representative of three independent experiments.
  • Figure 4B shows the frequency of CD8 + T cells recognizing the LF9-HLA-A24 complex in RCC17 TILs. Staining with HIV-HLA-A24 tetramer serves as a negative control. Data are representative of three independent experiments.
  • Figure 4C shows the frequency of CD8 + cells recognizing the LF9-HLA-A24 complex. PBMCs from healthy donors (HD) were stimulated in vitro with LF9 or an irrelevant peptide (GK12) for 14 days, and the frequency was measured by flow cytometry.
  • FIG. 4D shows a summary of the frequencies in different individuals (HD1, HD2, and HD3) after 14 days of stimulation with LF9 or GK12.
  • Figure 4E shows ELISPOT assays demonstrating IFN ⁇ production in HD PBMCs stimulated with LF9 or GK12 against T2-A24 cells pulsed with LF9 synthetic peptide or DMSO.
  • epitope peptide refers to a peptide that binds to MHC (HLA in humans), is presented as an antigen on the cell surface, and has antigenicity (can be recognized by T cells).
  • Epitope peptides include CTL epitope peptides, which are epitope peptides that are presented as an antigen by binding to MHC class I and recognized by CD8-positive T cells, and helper epitope peptides, which are epitope peptides that are presented as an antigen by binding to MHC class II and recognized by CD4-positive T cells.
  • tumor antigen peptides peptides derived from proteins that are specifically or excessively expressed in tumor cells are specifically referred to as tumor antigen peptides.
  • Antigen presentation refers to the phenomenon in which a peptide present in a cell binds to MHC, and this MHC/antigen peptide complex is localized on the cell surface.
  • antigens presented on the cell surface activate cellular and humoral immunity after being recognized by T cells, etc.
  • antigens presented on MHC class I activate cellular immunity and are recognized by the T cell receptors of naive T cells, inducing naive T cells to CTLs with cytotoxic activity. Therefore, as tumor antigen peptides used in immunotherapy, peptides that bind to MHC class I and are presented as antigens are preferred.
  • binding motif a characteristic of MHC.
  • HLA-A24 a type of human MHC
  • the binding motif of HLA-A24 is such that the second amino acid from the N-terminus is tyrosine, phenylalanine, methionine, or tryptophan, and the C-terminal amino acid is leucine, isoleucine, or phenylalanine.
  • motif substitution refers to a peptide in which a binding motif has been substituted with another binding motif. Those skilled in the art will naturally understand that in the present invention, motif substitutions also have the same effect as the peptide before substitution.
  • tumor includes benign and malignant tumors (cancer, malignant neoplasms). Cancer includes hematopoietic tumors, epithelial malignant tumors (carcinomas), and non-epithelial malignant tumors (sarcomas).
  • natural peptides of the present invention were isolated/identified by using the following method, which can comprehensively analyze the HLA-presented immune peptidome.
  • natural peptide refers to a peptide that is actually presented as an antigen on the cell surface.
  • natural antigen peptide refers to a natural peptide whose antigenicity has been confirmed.
  • the present inventors analyzed natural antigen peptides presented as antigens in human renal cell carcinoma (RCC) cells.
  • RCC human renal cell carcinoma
  • four types of peptides were identified as natural peptides presented exclusively as antigens in RCC17 tumor cells
  • three types of peptides were identified as natural peptides presented exclusively as antigens in RCC17 normal cells
  • one type of peptide was expressed in both tumor cells and normal cells.
  • five types of peptides were identified as natural peptides presented as antigens in RCC21 tumor cells, one of which was also presented exclusively as an antigen in RCC17 tumor cells.
  • hERV Human endogenous retroviruses
  • HERV Human endogenous retroviruses
  • hERV Gene Expression Products of the Present Invention
  • HERV Gene Expression Products of the Present Invention
  • a gene when a gene is simply referred to by its name, for example, "hERV” or "HERV”, it means a gene having a known nucleic acid sequence represented by that gene name, unless otherwise specified, and typically represents a cDNA or mRNA sequence, but is not limited to this as long as a person skilled in the art can recognize the sequence of the gene.
  • examples of preferred genes and their nucleic acid sequences in the present invention include the following genes represented by the sequences below.
  • the "hERV” gene of the present invention is a sequence corresponding to one ORF designated, for example, herv3895, herv2933, herv4024, herv1594 (Table 1 and FIG.
  • the nucleic acid sequence of herv3895 (6,407bb) is shown by SEQ ID NO:1, and its amino acid sequence is shown by SEQ ID NO:2. Therefore, the mRNA as the gene expression product of the present invention may be represented simply by the name of the gene.
  • protein when the term "protein” is added to the name of a gene, such as "hERV (derived) protein,” it means the protein encoded by the gene, its isoforms, and its homologs. Examples of the isoforms include splicing variants and variants such as SNPs based on individual differences.
  • examples include (1) proteins consisting of an amino acid sequence that has 90% or more, preferably 95% or more, and more preferably 98% or more homology with the protein encoded by the gene, and (2) proteins consisting of an amino acid sequence in which one or more, preferably 1 to several, and more preferably 1 to 10, 1 to 5, 1 to 3, or 1 or 2 amino acids have been substituted, deleted, added, or inserted in the amino acid sequence of the protein encoded by the gene.
  • Proteins that are preferred as gene expression products of the present invention include proteins that contain an amino acid sequence encoded by the above-mentioned genes (nucleic acid sequences), or proteins that have an amino acid sequence in which one to three, preferably one or two, amino acids have been substituted in the above-mentioned proteins. More preferred examples include proteins that have an amino acid sequence encoded by the above-mentioned genes (nucleic acid sequences).
  • the peptides of the present invention are partial peptides of a protein encoded by a hERV gene, and include peptides that bind to MHC, particularly HLA, preferably peptides that are antigen-presented by MHC, particularly HLA, and more preferably peptides that are antigen-presented by MHC, particularly HLA and can induce CTL.
  • MHC particularly HLA
  • HLA peptides that are antigen-presented by MHC
  • HLA preferably peptides that are antigen-presented by MHC, particularly HLA and can induce CTL.
  • HLA There are several types of HLA, and the peptides of the present invention are preferably capable of binding to HLA class I, and more preferably capable of binding to HLA-A24.
  • the peptides of the present invention may be subjected to processing or other treatments before binding to MHC, and peptides that generate epitope peptides as a result of such treatments are also included in the peptides of the present invention. Therefore, the amino acid length of the peptides of the present invention is not particularly limited as long as they have a sequence containing the amino acid sequence of the epitope peptide. However, it is preferable that the peptides of the present invention themselves are epitope peptides, and therefore the amino acid length is preferably about 8 to 14 amino acids, more preferably about 8 to 11 amino acids, and particularly preferably about 9 to 11 amino acids.
  • Epitope peptides that bind to HLA class I are about 8 to 14 amino acids long, preferably about 9 to 11 amino acids long, and are known to have a binding motif specific to the HLA they bind to in their sequence.
  • a peptide that binds to HLA-A02 has a binding motif in which the second amino acid from the N-terminus is leucine, isoleucine, or methionine, and/or the C-terminal amino acid is valine, leucine, or isoleucine
  • a peptide that binds to HLA-A24 has a binding motif in which the second amino acid from the N-terminus is tyrosine, phenylalanine, methionine, or tryptophan, and/or the C-terminal amino acid is leucine, isoleucine, or phenylalanine.
  • the peptide of the present invention comprises an epitope peptide, which is a partial peptide of a protein encoded by the hERV gene, consisting of 8 to 14 consecutive amino acids in the amino acid sequence of the protein, in which the second amino acid from the N-terminus is tyrosine, phenylalanine, methionine or tryptophan, and/or the C-terminal amino acid is leucine, isoleucine or phenylalanine, and more preferably is the epitope peptide itself.
  • epitope peptide which is a partial peptide of a protein encoded by the hERV gene, consisting of 8 to 14 consecutive amino acids in the amino acid sequence of the protein, in which the second amino acid from the N-terminus is tyrosine, phenylalanine, methionine or tryptophan, and/or the C-terminal amino acid is leucine, isoleucine or phenylalanine, and more preferably is the epi
  • epitope peptides consisting of any one of the amino acid sequences represented by LYDTVTHTF (SEQ ID NO: 3), PQTDQPREHLT (SEQ ID NO: 4), CNKTIYLLF (SEQ ID NO: 5), HFNSFHFL (SEQ ID NO: 6), TSRWSIPAL (SEQ ID NO: 7), SQYVFLTLQ (SEQ ID NO: 8), SFLMLSFQP (SEQ ID NO: 9), IFLRDRLLLF (SEQ ID NO: 10), KWFTVLDLK (SEQ ID NO: 11), KSACLYIFI (SEQ ID NO: 12), and LNLRLNSI (SEQ ID NO: 13).
  • the partial peptide includes an epitope peptide in which the second amino acid from the N-terminus is replaced with tyrosine, phenylalanine, methionine, or tryptophan, and/or the C-terminal amino acid is replaced with leucine, isoleucine, or phenylalanine, and more preferably is the epitope peptide itself.
  • epitope peptide in which the second amino acid from the N-terminus is replaced with tyrosine, phenylalanine, methionine, or tryptophan, and/or the C-terminal amino acid is replaced with leucine, isoleucine, or phenylalanine, in a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 3 to 13.
  • the peptide of the present invention may be modified at its N-terminus and/or C-terminus. Specific examples of such modifications include N-alkanoylation (e.g., acetylation), N-alkylation (e.g., methylation), C-terminal alkyl ester (e.g., ethyl ester), and C-terminal amide (e.g., carboxamide).
  • N-alkanoylation e.g., acetylation
  • N-alkylation e.g., methylation
  • C-terminal alkyl ester e.g., ethyl ester
  • C-terminal amide e.g., carboxamide
  • the in vivo activity of the peptides of the present invention can be confirmed by subjecting them to the CTL induction method described below or to an assay using a human model animal (WO 02/47474, Int J. Cancer: 100, 565-570 (2002)).
  • the peptide of the present invention further includes a peptide (polyepitope peptide) in which a plurality of epitope peptides, each of which contains at least one of the peptides of the present invention, are linked together.
  • a peptide polyepitope peptide
  • the polyepitope peptide having CTL inducing activity can also be exemplified as a specific example of the peptide of the present invention.
  • the polyepitope peptide of the present invention is (i) a peptide in which the peptide of the present invention (epitope peptide) and one or more CTL epitope peptides other than any peptide of the present invention are linked directly or via a suitable spacer; (ii) A peptide in which the peptide of the present invention and any one or more helper epitope peptides are linked directly or via a suitable spacer, or (iii) A peptide in which one or more helper epitope peptides are further linked to the polyepitope peptide described in (i) above, either directly or via a spacer as appropriate, and which is processed within antigen-presenting cells, and the resulting epitope peptide is presented to the antigen-presenting cells, thereby inducing CTL inducing activity.
  • the CTL epitope peptide other than the peptide of the present invention in (i) is not particularly limited, and specific examples thereof include epitope peptides derived from human ASB4, human OR7C1, and human DNAJB8 (e.g., peptides described in WO 2010/050190), epitope peptides derived from human FAM83B (WO 2015/050259), epitope peptides derived from human ASB4 (WO 2016/093243), and epitope peptides derived from human PVT1 (WO 2017/115798) (Patent Document 1).
  • epitope peptides derived from human ASB4, human OR7C1, and human DNAJB8 e.g., peptides described in WO 2010/050190
  • epitope peptides derived from human FAM83B WO 2015/050259
  • epitope peptides derived from human ASB4 WO 2016/093243
  • the spacer is not particularly limited as long as it does not adversely affect processing in antigen-presenting cells, and is preferably a linker that is linked to each epitope peptide by a peptide bond, such as a peptide linker in which several amino acids are linked, or a linker having an amino group and a carboxyl group at both ends.
  • a linker that is linked to each epitope peptide by a peptide bond
  • a linker in which several amino acids are linked
  • Specific examples include a glycine linker and a PEG (polyethylene glycol) linker.
  • An example of a glycine linker is polyglycine (e.g., a peptide consisting of six glycines; Cancer Sci, vol.
  • PEG linker is a linker derived from a compound having an amino group and a carboxyl group at both ends of PEG (e.g., H 2 N-(CH 2 ) 2-(OCH 2 CH 2 ) 3 -COOH; Angew. Chem. Int. Ed. 2008, 47, 7551-7556).
  • the epitope peptide of the present invention contained in the polyepitope peptide of the present invention may be one or more selected. That is, a plurality of the same epitope peptides may be linked, or a plurality of different epitope peptides may be linked. Of course, even when two or more epitope peptides are selected, one or more of the selected epitope peptides may be linked in a plurality of times. Similarly, a plurality of epitope peptides other than the peptide of the present invention may be linked to a plurality of epitope peptides.
  • the polyepitope peptide of the present invention may be one in which 2 to 12 epitope peptides are linked, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 epitope peptides are linked, and most preferably 2 epitope peptides are linked.
  • the epitope peptide linked to the peptide of the present invention is a helper epitope peptide
  • examples of the helper epitope peptide to be used include HBVc128-140 derived from hepatitis B virus and TT947-967 derived from tetanus toxin, etc.
  • the length of the helper epitope peptide can be about 13 to 30 amino acids, preferably about 13 to 17 amino acids.
  • polyepitope peptide can also be produced by a general peptide synthesis method as described above, or can be produced by ordinary DNA synthesis and genetic engineering techniques based on the sequence information of a polynucleotide encoding such a polyepitope peptide in which multiple epitope peptides are linked. That is, the polyepitope peptide can be produced by inserting the polynucleotide into a known expression vector, transforming a host cell with the resulting recombinant expression vector, culturing the resulting transformant, and recovering the polyepitope peptide having multiple epitopes of interest linked thereto from the culture. These techniques can be carried out according to the methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)) as mentioned above.
  • the polyepitope peptide produced as described above, in which multiple epitope peptides are linked, can be subjected to the above-mentioned in vitro assay or the in vivo assay using a human model animal described in WO 02/47474 and Int J. Cancer: 100, 565-570 (2002) (these documents are incorporated herein by reference) to confirm its CTL-inducing activity.
  • the peptides of the present invention (including polyepitope peptides) are useful for the prevention and/or treatment of cancer, and can be used as an active ingredient of a pharmaceutical composition.
  • the peptides of the present invention may be for the prevention and/or treatment of cancer.
  • the present invention also relates to the use of the peptides of the present invention in the manufacture of a medicament for the prevention and/or treatment of cancer.
  • polynucleotide of the present invention includes a polynucleotide encoding at least one of the peptides of the present invention.
  • the polynucleotide of the present invention may be any of cDNA, mRNA, cRNA, or synthetic DNA. It may also be in the form of a single strand or a double strand.
  • a polynucleotide consisting of a nucleotide sequence encoding an amino acid sequence that is a partial peptide of a protein encoded by a hERV gene and is predicted to have binding activity using MHC-peptide binding prediction programs such as BIMAS (http://www-bimas.cit.nih.gov/molbio/hla_bind/), SYFPEITHI (http://www.syfpeithi.de/), and IEDB (MHC-I processing predictions; http://www.iedb.org/), etc., may be mentioned.
  • MHC-peptide binding prediction programs such as BIMAS (http://www-bimas.cit.nih.gov/molbio/hla_bind/), SYFPEITHI (http://www.syfpeithi.de/), and IEDB (MHC-I processing predictions; http://www.iedb.org/), etc.
  • polynucleotides consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs: 3 to 13, and polynucleotides consisting of a nucleotide sequence encoding any two or more peptides selected from SEQ ID NOs: 3 to 13, or a polyepitope peptide in which a peptide selected from SEQ ID NOs: 3 to 13 and a helper epitope are linked, so as to be expressible.
  • the polynucleotide of the present invention can be either single-stranded or double-stranded.
  • a recombinant expression vector for expressing the peptide of the present invention can be prepared by inserting the polynucleotide of the present invention into an expression vector.
  • the category of the polynucleotide of the present invention also includes a recombinant expression vector prepared by inserting the double-stranded polynucleotide of the present invention into an expression vector.
  • the polynucleotide of the present invention is useful for the prevention and/or treatment of cancer, and can be an active ingredient of a pharmaceutical composition.
  • the polynucleotide of the present invention may be for the prevention and/or treatment of cancer. Furthermore, the present invention also relates to the use of the polynucleotide of the present invention in the manufacture of a medicament for the prevention and/or treatment of cancer.
  • expression vectors can be used in the present invention depending on the host and purpose, and those skilled in the art can select an appropriate one.
  • expression vectors that can be used in the present invention include plasmids, phage vectors, and virus vectors.
  • vectors include plasmid vectors such as pUC118, pUC119, pBR322, and pCR3, and phage vectors such as ⁇ ZAPII and ⁇ gt11.
  • vectors include pYES2 and pYEUra3.
  • vectors include pAcSGHisNT-A.
  • vectors include plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, and pRc/CMV, and virus vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors.
  • plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, and pRc/CMV
  • virus vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors.
  • the vector may appropriately have factors such as an inducible promoter, a gene encoding a signal sequence, a selection marker gene, and a terminator.
  • factors such as an inducible promoter, a gene encoding a signal sequence, a selection marker gene, and a terminator.
  • a sequence that is expressed as a fusion protein with thioredoxin, a His tag, or GST (glutathione S-transferase) or the like may be added.
  • a GST fusion protein vector such as pGEX4T having an appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc.) that functions in the host cell, a vector having a tag sequence such as Myc or His (such as pcDNA3.1/Myc-His), or even a vector that expresses a fusion protein with thioredoxin and a His tag (pET32a) can be used.
  • pGEX4T having an appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc.) that functions in the host cell
  • a vector having a tag sequence such as Myc or His
  • pcDNA3.1/Myc-His such as pcDNA3.1/Myc-His
  • pET32a a vector that expresses a fusion protein with thioredoxin and a His tag
  • a transformed cell containing the expression vector can be prepared by transforming a host with the expression vector prepared above. Therefore, the present invention includes a composition for gene transfer containing the expression vector.
  • any cell may be used as long as it does not impair the function of the polypeptide of the present invention, and examples thereof include Escherichia coli, yeast, insect cells, and animal cells.
  • Escherichia coli include E. coli K-12 strains HB101, C600, JM109, DH5 ⁇ , and AD494 (DE3).
  • yeast include Saccharomyces cerevisiae.
  • Examples of animal cells include L929 cells, BALB/c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, HeLa cells, and 293-EBNA cells.
  • Examples of insect cells include sf9.
  • the expression vector can be introduced into the host cell by a conventional method suitable for the host cell. Specific examples include the calcium phosphate method, the DEAE-dextran method, the electroporation method, and a method using a lipid for gene introduction (Lipofectamine, Lipofectin; Gibco-BRL). After the introduction, the cells are cultured in a conventional medium containing a selection marker, whereby transformed cells into which the expression vector has been introduced can be selected.
  • the peptide of the present invention can be produced by continuing to culture the transformed cells obtained as described above under suitable conditions.
  • the obtained peptide can be further isolated and purified by a general biochemical purification means.
  • the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography, etc.
  • the peptide of the present invention when expressed as a fusion protein with the above-mentioned thioredoxin, His tag, GST, etc., it can be isolated and purified by a purification method that utilizes the properties of these fusion proteins or tags.
  • the polynucleotides encoding the peptides of the present invention may be in the form of DNA or RNA.
  • polynucleotides of the present invention can be easily produced using conventional methods known in the art based on the amino acid sequence information of the peptides of the present invention and the sequence information of the DNA encoded thereby. Specifically, they can be produced by conventional DNA synthesis, PCR amplification, or the like.
  • the polynucleotides encoding the peptides of the present invention include polynucleotides encoding the epitope peptides.
  • CTL inducer/pharmaceutical composition containing the peptide of the present invention as an active ingredient
  • the peptide of the present invention has CTL inducing activity and can be a CTL inducer as a tumor antigen peptide.
  • the present inventors have discovered for the first time that the protein encoded by the hERV gene is a tumor antigen, and that a peptide derived from the protein binds to an HLA class I antigen on the surface of a tumor cell to form a complex, which is delivered to the cell surface and presented as an antigen. Therefore, the protein encoded by the hERV gene itself can also be a CTL inducer.
  • peripheral blood lymphocytes are isolated from a human positive for HLA-A24 antigen, and stimulated in vitro with the peptide of the present invention and/or a protein encoded by the hERV gene to induce CTLs that specifically recognize HLA-A24 antigen-positive cells pulsed with the peptide (J.Immunol., 154, p2257, 1995).
  • the presence or absence of induction of CTLs can be confirmed, for example, by measuring the amount of various cytokines (e.g., IFN- ⁇ ) produced by CTLs in response to antigen peptide-presenting cells, for example, by ELISA.
  • CTL clones can be established by the methods described in Int. J. Cancer, 39, 390-396, 1987; N. Eng. J. Med, 333, 1038-1044, 1995, and the like.
  • CTLs induced by the peptide of the present invention and/or the protein encoded by the hERV gene have a cytotoxic effect against cells presenting epitope peptides derived from the peptide of the present invention and/or other proteins encoded by the hERV gene as antigens, and have the ability to produce lymphokines.
  • the peptide of the present invention is a tumor antigen peptide
  • the protein encoded by the hERV gene is degraded in cells to produce tumor antigen peptides, and thus can exert an antitumor effect, preferably an anticancer effect, through these functions.
  • the peptide of the present invention and/or the protein encoded by the hERV gene, and the CTLs induced thereby can be used as active ingredients of medicines and pharmaceutical compositions for preventing and/or treating cancer.
  • a CTL inducer containing as an active ingredient the peptide of the present invention and/or a protein encoded by the hERV gene is administered to a cancer patient, an epitope peptide derived from the peptide of the present invention and/or the protein encoded by the hERV gene is presented to the HLA antigen, preferably the HLA-A24 antigen, of antigen-presenting cells, and CTLs that specifically recognize the complex between the HLA antigen and the presented peptide proliferate and can destroy cancer cells, resulting in the prevention and/or treatment of cancer.
  • the CTL inducer containing as an active ingredient the peptide of the present invention and/or the protein encoded by the hERV gene can be preferably used for subjects who are HLA-A24 antigen-positive and suffering from hERV-positive cancer.
  • hERV-positive cancers include gastrointestinal cancer (anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer), breast cancer, uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin cancer, lung cancer, colon cancer, brain cancer, heart cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer, spleen cancer, thymic cancer, prostate cancer, ovarian cancer, and blood cancer, and the CTL inducer of the present invention can be used for the prevention and/or treatment of these cancers.
  • gastrointestinal cancer anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer
  • breast cancer uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin
  • prevention of cancer includes not only preventing patients from contracting cancer, but also preventing recurrence in patients who have had their primary tumor removed by surgery, and preventing metastasis of tumors that could not be completely removed by cancer treatments such as surgery, radiation therapy or chemotherapy.
  • treatment of cancer includes not only curing cancer or improving symptoms by shrinking the cancer, but also preventing progression by suppressing the proliferation of cancer cells, the expansion of tumors, or the metastasis of cancer cells from the primary tumor.
  • a CTL inducer containing as an active ingredient the peptide of the present invention and/or a protein encoded by the hERV gene is particularly effective for HLA-A24-positive cancer patients suffering from hERV-positive cancer.
  • it can be used for the prevention or treatment of, for example, gastrointestinal cancer (anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer), breast cancer, uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin cancer, lung cancer, colon cancer, brain cancer, heart cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer, spleen cancer, thymus cancer, prostate cancer, ovarian cancer, and blood cancer.
  • gastrointestinal cancer anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pan
  • a pharmaceutical composition containing as an active ingredient the peptide of the present invention and/or a protein encoded by the hERV gene is also included in the present invention.
  • a pharmaceutical composition is preferably a composition for preventing and/or treating cancer, i.e., a cancer preventive and/or therapeutic agent.
  • the pharmaceutical composition of the present invention prevents and/or treats cancer by inducing cancer cell-specific CTLs, i.e., activating cancer cell-specific cellular immunity, it is preferably a vaccine for preventing and/or treating cancer.
  • pseudouridine can be used instead of uridine to suppress inflammatory responses.
  • compositions containing the peptide of the present invention as an active ingredient may contain a single CTL epitope (the peptide of the present invention) as an active ingredient, or may contain a polyepitope peptide in which the peptide is linked to other peptides (CTL epitopes or helper epitopes).
  • CTL epitopes or helper epitopes polyepitope peptides in which multiple CTL epitopes (antigenic peptides) are linked have been shown to have efficient CTL induction activity in vivo.
  • the polyepitope peptides When administered in the form of such polyepitope peptides, the polyepitope peptides are taken up into antigen-presenting cells, and then the individual antigen peptides generated by intracellular degradation bind to HLA antigens to form complexes, which are then presented at high density on the surface of the antigen-presenting cells, and CTLs specific to these complexes proliferate efficiently in the body and destroy cancer cells. In this way, cancer treatment or prevention is promoted.
  • composition containing as an active ingredient the peptide of the present invention and/or the protein encoded by the hERV gene can be administered in a mixture with a medicament-acceptable carrier, such as a suitable adjuvant, or in combination with the peptide, so that cellular immunity is effectively established.
  • a medicament-acceptable carrier such as a suitable adjuvant
  • any adjuvant known in the art such as those described in the literature (e.g., Clin Infect Dis.: S266-70, 2000), can be used.
  • the adjuvant include, for example, aluminum hydroxide, aluminum phosphate, and calcium phosphate as gel types, CpG, monophosphoryl lipid A (MPL), cholera toxin, Escherichia coli heat labile toxin, pertussis toxin, and muramyl dipeptide (MDP) as bacterial cell types, Freund's incomplete adjuvant, MF59, and SAF as oil emulsion types (emulsion preparations), immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres, and saponin-derived QS-21 as polymer nanoparticle types, and nonionic block copolymers, muramyl peptide analogs, and synthetic types.
  • MPL monophosphoryl lipid A
  • cholera toxin Escherichia coli heat labile
  • the dosage form of the CTL inducer/pharmaceutical composition containing as an active ingredient the peptide of the present invention and/or a protein encoded by the hERV gene is not particularly limited, and examples thereof include an oil emulsion (emulsion preparation), a polymer nanoparticle, a liposome preparation, a particulate preparation bound to beads having a diameter of several ⁇ m, a lipid-bound preparation, a microsphere preparation, and a microcapsule preparation.
  • the administration method may be any known administration method such as intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, etc.
  • the dose of the peptide of the present invention in the formulation can be appropriately adjusted depending on the disease to be treated, the age and body weight of the patient, etc., but is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 10 mg, and is preferably administered once every few days to several months.
  • CTL inducer/pharmaceutical composition containing the polynucleotide of the present invention as an active ingredient
  • Cells expressing the polynucleotide of the present invention and/or a polynucleotide encoding the hERV protein are capable of presenting other epitope peptides derived from the peptide of the present invention and/or the protein encoded by the hERV gene as antigens, and are therefore characterized in that they are recognized by T cells via T cell receptors. Therefore, the polynucleotide of the present invention and/or a polynucleotide encoding the hERV protein can also be an inducer of CTL.
  • the tumor antigen peptide when the polynucleotide of the present invention and/or a polynucleotide encoding a hERV protein incorporated in an expression vector is administered to a cancer patient by the following method, the tumor antigen peptide is highly expressed in the antigen-presenting cells. The resulting tumor antigen peptide then binds to an HLA antigen such as HLA-A24 antigen to form a complex, and the complex is presented at a high density on the surface of the antigen-presenting cells, causing cancer-specific CTLs to efficiently proliferate in the body and destroy the cancer cells. In this way, cancer treatment or prevention is achieved.
  • HLA antigen such as HLA-A24 antigen
  • compositions containing the polynucleotide of the present invention and/or a polynucleotide encoding a hERV protein are also included in the present invention.
  • Such pharmaceutical compositions are preferably compositions for preventing and/or treating cancer, i.e., preventive and/or therapeutic agents for cancer.
  • the pharmaceutical composition of the present invention prevents and/or treats cancer by inducing CTLs specific to cancer cells (preferably cancer stem cells), i.e., activating cancer cell-specific cellular immunity, and is therefore preferably a vaccine for preventing and/or treating cancer.
  • the CTL inducer/pharmaceutical composition containing the polynucleotide of the present invention as an active ingredient can be preferably used for HLA-A24 antigen-positive subjects suffering from hERV-positive cancer.
  • hERV-positive cancer include gastrointestinal cancer (anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer), breast cancer, uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin cancer, lung cancer, colorectal cancer, brain cancer, heart cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer, spleen cancer, thymus cancer, prostate cancer, ovarian cancer, and blood cancer, and the CTL inducer of the present invention can be used for the prevention or treatment of these cancers.
  • Examples of the method using a viral vector include a method of incorporating the DNA of the present invention into a DNA or RNA virus such as a retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, Simbis virus, etc.
  • a method using a retrovirus, adenovirus, adeno-associated virus, vaccinia virus, etc. is particularly preferred.
  • DNA vaccine method a method in which an expression plasmid is directly administered intramuscularly
  • liposome method the lipofectin method
  • microinjection method the calcium phosphate method
  • electroporation method etc.
  • the in vivo method is more preferable.
  • an appropriate administration route and administration form may be appropriately selected according to the disease, symptoms, etc. to be treated.
  • it may be administered in a form that can be injected into a vein, an artery, subcutaneously, intradermally, intramuscularly, etc.
  • it may take the form of a preparation such as a liquid, but is generally an injection containing the polynucleotide of the present invention as an active ingredient, and a medicamentically acceptable carrier (carrier) may be added as necessary.
  • liposomes or membrane-fusogenic liposomes (Sendai virus (HVJ)-liposomes, etc.) containing the polynucleotide of the present invention may be in the form of a liposome preparation such as a suspension, a cryoagent, or a centrifugally concentrated cryoagent.
  • a liposome preparation such as a suspension, a cryoagent, or a centrifugally concentrated cryoagent.
  • the content of the polynucleotide of the present invention in the formulation can be appropriately adjusted depending on the disease to be treated, the age and body weight of the patient, etc., but it is usually preferable to administer a polynucleotide content of 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg, of the polynucleotide of the present invention once every few days to several months.
  • a polynucleotide content of 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg, of the polynucleotide of the present invention once every few days to several months.
  • Those skilled in the art can appropriately select suitable cells, vectors, administration methods, administration forms and dosages.
  • polynucleotides encoding polyepitope peptides in which multiple CTL epitopes (tumor antigen peptides) are linked, or polynucleotides encoding polyepitope peptides in which CTL epitopes and helper epitopes are linked, have efficient CTL inducing activity in vivo.
  • the pharmaceutical composition of the present invention exerts an antitumor effect by inducing tumor-specific immune cells, it can exert a higher therapeutic effect by simultaneously suppressing the function of immune checkpoints. Therefore, in a preferred embodiment, the pharmaceutical composition of the present invention is used together with an immune checkpoint inhibitor.
  • any agent known as an immune checkpoint inhibitor can be used as long as it does not inhibit the ability of the composition of the present invention to induce CTLs.
  • Known immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-B7-H5 antibodies, and anti-TIGIT antibodies.
  • Antigen-presenting cells of the present invention The peptide and polynucleotide of the present invention described above can be used in vitro, for example, as follows. That is, by contacting either the peptide or polynucleotide of the present invention with a cell having antigen-presenting ability in vitro, an antigen-presenting cell that presents the antigen peptide of the present invention as an antigen can be prepared. Therefore, in one aspect of the present invention, an antigen-presenting cell that presents a complex of an HLA antigen, preferably an HLA-A24 antigen, and the peptide of the present invention on the cell surface, and a method for producing the same are provided.
  • the peptide and polynucleotide of the present invention can be used to prevent and/or treat cancer. Therefore, the antigen-presenting cell or the method for producing the same of this aspect preferably uses cells isolated from a cancer patient. Specifically, an antigen-presenting cell that presents a complex of an HLA antigen, preferably an HLA-A24 antigen, and the peptide of the present invention on the cell surface is produced by contacting either the peptide or polynucleotide of the present invention with an isolated cell having antigen-presenting ability derived from a cancer patient in vitro.
  • an HLA antigen preferably an HLA-A24 antigen
  • the "cells having antigen-presenting ability” are not particularly limited as long as they express MHC, preferably HLA, and more preferably HLA-A24 antigen on the cell surface and are capable of presenting the peptide of the present invention.
  • professional antigen-presenting cells are preferred, and dendritic cells, which are considered to have particularly high antigen-presenting ability, are more preferred.
  • the substance added to prepare the antigen-presenting cells of the present invention from the cells having antigen-presenting ability may be either the peptide or polynucleotide of the present invention.
  • the antigen-presenting cells of the present invention can be obtained, for example, by isolating cells having antigen-presenting ability from a cancer patient, pulsing the cells with the peptide of the present invention in vitro, and allowing the cells to present a complex of HLA-A24 antigen and the peptide of the present invention (Cancer Immunol. Immunother., 46:82, 1998; J. Immunol., 158, p1796, 1997; Cancer Res., 59, p1184, 1999).
  • dendritic cells for example, lymphocytes are separated from peripheral blood of a cancer patient by the Ficoll method, non-adherent cells are then removed, and the adherent cells are cultured in the presence of GM-CSF and IL-4 to induce dendritic cells, which are then cultured with the peptide of the present invention and pulsed, thereby preparing the antigen-presenting cells of the present invention.
  • the polynucleotide when preparing the antigen-presenting cells of the present invention by introducing the polynucleotide of the present invention into the cells having the antigen-presenting ability, the polynucleotide may be in the form of DNA or RNA.
  • DNA this can be done with reference to Cancer Res., 56: p. 5672, 1996 and J. Immunol., 161: p. 5607, 1998 (these documents are incorporated herein by reference)
  • RNA this can be done with reference to J. Exp. Med., 184: p. 465, 1996 (these documents are incorporated herein by reference), etc.
  • the antigen-presenting cells can be used as an active ingredient of a CTL inducer and/or pharmaceutical composition.
  • the CTL inducer and/or pharmaceutical composition containing the antigen-presenting cells as an active ingredient preferably contains physiological saline, phosphate-buffered saline (PBS), culture medium, etc., in order to stably maintain the antigen-presenting cells.
  • administration methods include intravenous administration, subcutaneous administration, and intradermal administration.
  • CTLs specific to cancer cells that present the peptide of the present invention as an antigen are efficiently induced in the body of a patient suffering from hERV-positive cancer, and as a result, hERV-positive cancer that presents the peptide of the present invention as an antigen can be prevented and/or treated.
  • Cytotoxic T cells (CTL) of the present invention can be used in vitro, for example, as follows. That is, CTLs can be induced by contacting peripheral blood lymphocytes with either the peptides or polynucleotides of the present invention in vitro. Thus, in one aspect of the present invention, CTLs that specifically damage cells that present the peptides of the present invention as antigens and a method for inducing the same are provided. As described above, the peptides and polynucleotides of the present invention can be used to prevent and/or treat cancer.
  • the CTLs and the method for inducing the same of this aspect preferably use peripheral blood lymphocytes derived from cancer patients.
  • CTLs that specifically damage cells that present the peptides of the present invention as antigens are induced by contacting peripheral blood lymphocytes derived from cancer patients in vitro with either the peptides or polynucleotides of the present invention.
  • melanoma adoptive immunotherapy, in which a patient's own tumor-infiltrating T cells are cultured in large quantities outside the body and then returned to the patient, has been shown to be effective (J. Natl. Cancer. Inst., 86:1159, 1994).
  • metastasis has been suppressed by stimulating spleen cells in vitro with the tumor antigen peptide TRP-2, proliferating CTLs specific to the tumor antigen peptide, and administering the CTLs to mice transplanted with melanoma (J. Exp. Med., 185:453, 1997).
  • the CTLs can be used as an active ingredient of a cancer treatment or prevention agent.
  • the treatment or prevention agent preferably contains physiological saline, phosphate-buffered saline (PBS), culture medium, etc., in order to stably maintain the CTLs.
  • Methods of administration include intravenous administration, subcutaneous administration, and intradermal administration.
  • the CTL of the present invention can exert cytotoxic activity by targeting the complex of the peptide of the present invention and HLA that is presented as an antigen on tumor cells. That is, the T cell receptor (TCR) of the CTL of the present invention recognizes the complex of the peptide of the present invention and HLA.
  • TCR T cell receptor
  • adoptive immunotherapy has been devised in which a TCR gene that recognizes a specific peptide-HLA complex expressed in CTL is cloned, the TCR gene is introduced into CD8 + T cells collected from a cancer patient to artificially produce CTLs, and the CTLs are cultured in large quantities and then returned to the patient (e.g., Ochi et al., Blood.
  • the term "artificial CTL” refers to a CTL produced by introducing a gene encoding a TCR that recognizes a complex of a peptide and HLA into a T cell, as described above, and this can also be used for cancer treatment in the same way as the above-mentioned natural CTL. Therefore, such artificial CTLs are also included in the CTL of the present invention.
  • the TCR that recognizes the complex of the peptide of the present invention and HLA and is genetically introduced into the artificial CTL may be appropriately modified to increase the binding affinity to the complex or the cytotoxic activity.
  • the "artificial CTL” also includes a CTL prepared by appropriately modifying a gene encoding a TCR that recognizes the complex of the peptide of the present invention and HLA, and then genetically introducing the gene into a T cell derived from a patient.
  • the artificial CTL can be prepared by a method known in the art.
  • Tumor-specific CTL detection agent using the peptide of the present invention can be recognized by tumor-specific CTL, and is therefore useful as a component of a tumor-specific CTL detection agent.
  • the present invention also relates to a tumor-specific CTL detection agent comprising the peptide of the present invention.
  • the tumor-specific CTL detection agent of the present invention comprises an HLA multimer (monomer, dimer, tetramer, pentamer, and dextramer) comprising the peptide of the present invention and HLA-A24.
  • HLA tetramer refers to a tetramer formed by biotinylating a complex (HLA monomer) in which the HLA ⁇ chain and ⁇ 2 microglobulin are associated with a peptide (epitope peptide) and binding it to avidin (Science 279: 2103-2106 (1998), Science 274: 94-96 (1996)).
  • HLA tetramers containing various antigen peptides are commercially available (for example, from Medical and Biological Laboratories, Inc.), and HLA tetramers containing the peptide of the present invention and HLA-A24 can be easily prepared.
  • HLA dimers and HLA pentamers are also based on the same principle, and in these, the HLA monomer is dimerized and pentamed, respectively. Therefore, HLA multimers containing the peptide of the present invention and HLA-A24 are also an aspect of the present invention.
  • HLA tetramers that contain a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 13 and HLA-A24.
  • the HLA tetramer is preferably fluorescently labeled so that bound CTLs can be easily selected or detected by known detection means such as flow cytometry or a fluorescent microscope.
  • Specific examples include HLA tetramers labeled with phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), etc.
  • HLA-A24 ⁇ -chain expression vector and ⁇ 2 microglobulin expression vector are introduced into Escherichia coli or mammalian cells capable of expressing proteins, and are expressed.
  • Escherichia coli e.g., BL21
  • the resulting monomeric HLA-A24 is mixed with the peptide of the present invention to form a soluble HLA-peptide complex.
  • the sequence at the C-terminal site of the ⁇ -chain of HLA-A24 in the HLA-peptide complex is biotinylated with BirA enzyme.
  • an HLA tetramer can be prepared.
  • the present inventors have found that the hERV gene is a tumor antigen that is specifically and highly expressed in tumor tissues of renal cell carcinoma. That is, the present inventors have clarified that the hERV gene is a gene that is highly expressed in tumor cells of renal cell carcinoma, while its expression is observed to be slight in normal cells of the testis, pancreas, spleen, and kidney. From such findings, it has been found that the hERV gene can be used as a marker for identifying tumor cells, particularly cancer cells. Therefore, in one aspect, the present invention relates to a tumor cell detection agent, including a detection agent for detecting an expression product of the hERV gene.
  • hERV means the hERV gene, etc.
  • the hERV gene is preferably a human gene, but may be a homolog thereof.
  • gene expression refers to a series of biological reactions starting from the transcription of the gene
  • expression product refers to a molecule produced by this series of biological reactions, such as mRNA or an endogenous polypeptide.
  • the endogenous polypeptide, which is the expression product of a gene is preferably a protein that is ultimately produced by the expression of the gene.
  • the term "detection agent for a gene expression product” refers to an agent for qualitatively and/or quantitatively detecting an expression product of the hERV gene.
  • the tumor cell detection agent of the present invention includes a detection agent for detecting the expression product of the hERV gene.
  • a detection agent for detecting the expression product of the hERV gene When the expression product of the hERV gene is detected in the detection target, it can be determined that the detection target has tumor cells, i.e., tumor cells have been detected.
  • the tumor cell detection agent of the present invention can be used in vivo or in vitro, but is preferably used in vitro on a cell population (detection target) derived from a biological sample collected from an individual organism (test target).
  • the detection of tumor cells in the cell population derived from the biological sample that is the detection target means that tumor cells are also detected in the individual organism from which the biological sample that is the test target was collected, i.e., the individual organism has tumor cells.
  • a method for detecting tumor cells in an individual organism using the tumor cell detection agent of the present invention i.e., a method for testing whether the individual organism has a tumor
  • the organism to be tested may be any organism capable of having a tumor, but is preferably a human or non-human mammal (e.g., rodents such as mice, rats, guinea pigs, hamsters, etc.; primates such as chimpanzees; even-toed ungulates such as cows, goats, sheep, etc.; odd-toed ungulates such as horses, rabbits, dogs, cats, etc.), and more preferably a human.
  • rodents such as mice, rats, guinea pigs, hamsters, etc.
  • primates such as chimpanzees
  • even-toed ungulates such as cows, goats, sheep, etc.
  • odd-toed ungulates such as horses, rabbits, dogs, cats, etc.
  • the cell population to be detected can be a cell population derived from any biological sample obtained from the above-mentioned test subject, but is preferably a cell population derived from a biological sample obtained from a human, and more preferably a cell population containing cells derived from one or more biological samples selected from the group consisting of tissues in which it has been confirmed that the hERV gene is hardly expressed in the cells, such as the gastrointestinal tract (anus, bile duct, colon, esophagus, gallbladder, gastrointestinal stroma, liver, pancreas, rectum, small intestine, and stomach), breast, uterus, soft tissue, bladder, head and neck, skin, lung, large intestine, brain, heart, placenta, skeletal muscle, kidney, spleen, thymus, prostate, ovary, and blood.
  • the gastrointestinal tract anus, bile duct, colon, esophagus, gallbladder, gastrointestinal stroma, liver, pancreas,
  • the detection agent for the expression product of the hERV gene contained in the tumor cell detection agent of the present invention may vary depending on the expression product to be detected, and a person skilled in the art may select the most suitable one as appropriate.
  • the expression product is mRNA
  • any mRNA detection method known in the art may be used, and examples include, but are not limited to, RT-PCR, in situ hybridization, Northern blotting, real-time RT-PCR, etc., of which RT-PCR is preferred due to its high detection sensitivity and ease of experimental procedures.
  • examples include, but are not limited to, Western blotting, immunohistochemical staining, etc.
  • the detection agent for the expression product of the hERV gene used may vary depending on the expression product to be detected and the detection method employed, and a person skilled in the art may select the most suitable one as appropriate.
  • the expression product to be detected may be a single expression product or a combination of multiple expression products.
  • the peptide of the present invention is presented as an antigen by tumor cells as a CTL epitope peptide.
  • the peptide of the present invention or an antibody that recognizes the complex of the peptide and MHC can be used as a tumor marker.
  • examples of such antibodies include an antibody (preferably a monoclonal antibody) that specifically recognizes the peptide of the present invention, and a TCR (T cell antigen receptor)-like antibody that recognizes the complex of the peptide of the present invention and HLA, preferably the complex of HLA-A24.
  • the present invention also relates to an antibody that recognizes the peptide of the present invention or the complex of the peptide and MHC, particularly a monoclonal antibody or a T cell antigen receptor-like antibody.
  • a "TCR-like antibody” is a molecule having a TCR-like binding ability (antigen recognition ability) to a complex (pMHC) of a peptide derived from a fragmented antigen and a major histocompatibility complex (MHC) molecule.
  • TCR-like antibody is a molecule having a TCR-like binding ability (antigen recognition ability) to a complex (pMHC) of a peptide derived from a fragmented antigen and a major histocompatibility complex (MHC) molecule.
  • a TCR-like antibody that recognizes a complex of a peptide derived from a tumor antigen and MHC can recognize cancer cells that present a tumor antigen peptide that can be targeted by CTL, dendritic cells that phagocytose cancer cells and present a tumor antigen peptide on MHC class I, etc.
  • the TCR-like antibodies can be produced by the methods described in, for example, Eur J Immunol. 2004;34:2919-29.
  • complex-specific antibodies can be obtained by immunizing an animal such as a mouse with an MHC and peptide complex. It is also possible to obtain complex-specific antibodies using the phage display method.
  • the present invention also relates to a tumor detection agent containing the above-mentioned TCR-like antibody. Furthermore, since the peptide of the present invention is presented not only by tumor cells but also by antigen-presenting cells, particularly professional antigen-presenting cells such as dendritic cells, the above-mentioned TCR-like antibody is also useful for detecting antigen-presenting cells presenting the peptide of the present invention.
  • the term "antibody” includes not only immunoglobulin molecules but also functional fragments of antibodies such as Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, and sc(Fv)2. Multimers of these functional fragments (e.g., dimers, trimers, tetramers, polymers) are also included in the antibodies of the present invention.
  • the peptide of the present invention is presented by tumor cells as a CTL epitope peptide, and therefore a TCR-like antibody that recognizes the peptide of the present invention and/or a complex between the peptide and HLA, preferably a complex between the peptide and HLA-A24, can bind to the complex present on the cell surface in a subject.
  • the TCR-like antibody binds to the tumor cell surface
  • the Fc receptor of effector cells such as macrophages and NK cells binds to the Fc portion of the antibody, and the effector cells attack tumor cells, generating antibody-dependent cellular cytotoxicity (ADCC) activity, thereby treating the tumor. Therefore, the above TCR-like antibody is also useful for preventing and/or treating cancer. Therefore, the present invention also relates to a preventive and/or therapeutic agent for cancer, comprising the TCR-like antibody of the present invention.
  • bispecific antibodies have been developed in which two antigen-binding sites have been modified so that each binds to a different antigen.
  • Bispecific antibodies recognize cancer cell surface antigens such as MHC-antigen peptide complexes at one antigen-binding site and lymphocyte surface antigens such as CD3 at the other antigen-binding site, making it possible to bind and accumulate cells bearing lymphocyte surface antigens, such as CTLs and effector cells, in the vicinity of cancer cells.
  • Lymphocytes that are bound in the vicinity of cancer cells not only exhibit antitumor activity such as ADCC activity, but also attack cancer cells by exerting a bystander effect that activates naive immune cells around the cancer cells to have antitumor properties by secreting cytokines, etc.
  • the present invention therefore also encompasses bispecific antibodies that specifically recognize the peptide of the present invention and/or a complex of said peptide with HLA, and a lymphocyte surface antigen.
  • the lymphocyte surface antigen that is specifically recognized is not particularly limited as long as it is an antigen that is specifically expressed on the surface of lymphocytes, but preferred examples include CD3, CD16, CD64, and the like.
  • CD3 is a cell surface antigen involved in inducing the cytotoxic activity of CTLs, and when an antibody binds to CD3, it is possible to activate CTLs in an HLA-nonrestricted manner without recognizing the HLA-cancer antigen complex, and it is expected that potent cytotoxic activity will be exerted, making it preferable.
  • a new immune cell therapy has been devised in which a chimeric antigen receptor (CAR) modified by genetically engineering a part of a monoclonal antibody specific to a tumor antigen is genetically introduced into T cells derived from a patient, and the genetically modified T cells are expanded and cultured ex vivo and then transfused back to the patient (Nat Rev Immunol. 2012;12:269-81).
  • CAR chimeric antigen receptor
  • peripheral blood mononuclear cells collected from a patient are cultured in the presence of an anti-CD3 antibody and IL-2 or the like to activate the T cells, and then a gene encoding the CAR is introduced into the T cells using a transformation vector such as a retrovirus vector or lentivirus vector to produce genetically modified T cells.
  • a transformation vector such as a retrovirus vector or lentivirus vector to produce genetically modified T cells.
  • a "chimeric antigen receptor” is a chimeric protein molecule designed to have a single-chain antibody (scFv) at the N-terminus, in which the light chain and heavy chain of an antibody variable region of an antibody that recognizes a molecule present on the cell surface of a cancer cell are linked in series, and a CD3 ⁇ chain of a molecule constituting a T cell receptor (TCR)/CD3 complex at the C-terminus.
  • TCR T cell receptor
  • one or more costimulatory molecules may be incorporated between the scFv and the ⁇ chain.
  • a CAR can be produced using the TCR-like antibody of this embodiment (including an antibody molecule or a fragment thereof that can be designed from a TCR-like antibody) as the scFv.
  • the present invention also relates to a preventive and/or therapeutic agent for cancer, comprising genetically modified T cells or artificial CTLs into which a CAR that recognizes a complex between a peptide derived from a tumor antigen and MHC of the present invention has been introduced.
  • Tumor detection method (test method, diagnostic method)
  • the present invention provides a method for detecting a tumor (testing method, diagnostic method) that utilizes the above-mentioned CTL detecting agent or tumor cell detecting agent (tumor detecting agent) of the present invention.
  • the detection method (diagnosis method) of the present invention using the CTL detection agent of the present invention typically involves collecting blood from a subject or collecting a portion of a test tissue suspected of having a tumor by biopsy or the like, and detecting and measuring the amount of CTLs contained therein that recognize a complex of a hERV-derived tumor antigen peptide and an HLA antigen using the CTL detection agent of the present invention, thereby detecting, examining, or diagnosing the presence or absence, or the degree of, hERV-positive cancer (tumor), such as gastrointestinal cancer (anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer), breast cancer, uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin cancer, lung cancer, colorectal cancer, brain cancer, cardiac cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer
  • the detection method (testing method, diagnostic method) of the present invention using the tumor detection agent of the present invention typically involves taking blood from a subject or taking a portion of a test tissue suspected of having a tumor by biopsy or the like, and detecting and measuring the amount of hERV expression product contained therein using the tumor detection agent of the present invention, thereby detecting, testing, or diagnosing the presence or absence, or the severity, of hERV-positive cancer (tumor) such as gastrointestinal cancer (anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer), breast cancer, uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin cancer, lung cancer, colorectal cancer, brain cancer, cardiac cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer, spleen cancer, thymic cancer, prostate cancer, ovarian cancer, and blood cancer
  • the detection method (testing method, diagnostic method) of the present invention using the tumor detection agent of the present invention typically involves collecting blood from a subject or collecting a portion of a test tissue suspected of having a tumor by biopsy or the like, and detecting and measuring the amount of cells presenting a complex of a hERV-derived tumor antigen peptide and an HLA antigen contained therein using the tumor detection agent of the present invention, thereby detecting, testing, or diagnosing the presence or absence, or the degree of, hERV-positive cancer (tumor), such as gastrointestinal cancer (anal cancer, bile duct cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, and stomach cancer), breast cancer, uterine cancer, soft tissue tumor, bladder cancer, head and neck cancer, skin cancer, lung cancer, colon cancer, brain cancer, cardiac cancer, placental cancer, tumors derived from skeletal muscle, kidney cancer, spleen cancer
  • the detection (testing, diagnosis) method of the present invention can also detect (test, diagnose) the presence or absence of improvement of a tumor or the degree of improvement of the tumor when, for example, a therapeutic drug is administered to improve the tumor in a patient having the tumor. Furthermore, the detection (testing, diagnosis) method of the present invention can also be used to select patients to whom a drug containing the peptide or polynucleotide of the present invention as an active ingredient can be effectively applied, and to predict or determine the therapeutic effect of the drug.
  • tumor detection agent of the present invention it is possible to detect cancer cells presenting tumor antigen peptides that can actually be targeted by CTLs induced in the patient's body by administering a cancer vaccine containing the peptide of the present invention as an active ingredient.
  • a specific embodiment of the detection (test) method of the present invention using the CTL detection agent of the present invention comprises the following steps (a) and (b), and optionally (c): (a) contacting a biological sample obtained from a subject with the CTL detection agent of the present invention; (b) measuring the amount of CTLs that recognize the complex of the hERV-derived tumor antigen peptide and the HLA antigen in the biological sample, using the amount of cells bound to the CTL detection agent as an index; (c) determining the presence or absence of cancer based on the results of (b).
  • a specific embodiment of the diagnostic method of the present invention using the CTL detection agent of the present invention comprises the above steps (a), (b) and (c).
  • a specific embodiment of the detection (examination) method of the present invention using the tumor cell detecting agent of the present invention comprises the following steps (d) and (e), and optionally (f): (d) contacting a biological sample obtained from a subject with the tumor cell detection agent of the present invention; (e) measuring the amount of hERV expression product in the biological sample; (f) A step of determining the presence or absence of cancer based on the results of (e).
  • a specific embodiment of the diagnostic method of the present invention using the tumor cell detecting agent of the present invention comprises the above steps (d), (e) and (f).
  • An embodiment of the method for detecting tumor cells using the tumor cell detecting agent of the present invention includes the following step (f') instead of the above steps (d), (e) and (f): (f') determining the presence or absence of tumor cells in the biological sample based on the results of (e).
  • the biological sample used herein may be a sample prepared from a subject's biological tissue (such as a tissue suspected of containing cancer cells and its surrounding tissue, or blood). Specifically, the biological sample may be a sample containing tissue cells collected from the tissue.
  • a specific embodiment of the detection (test) method of the present invention using the tumor detecting agent of the present invention comprises the following steps (g) and (h), and optionally (i): (g) contacting a biological sample obtained from a subject with the tumor detecting agent of the present invention; (h) measuring the amount of cells presenting a complex of a hERV-derived tumor antigen peptide and an HLA antigen in the biological sample using the amount of cells bound to the tumor detection agent as an index; (i) A step of determining the presence of cancer based on the results of (h).
  • a specific embodiment of the diagnostic method of the present invention using the tumor detecting agent of the present invention comprises the above steps (g), (h) and (i).
  • the biological sample used herein may be a sample prepared from a subject's biological tissue (such as a tissue suspected of containing cancer cells and its surrounding tissue, or blood). Specifically, the biological sample may be a sample containing tissue cells collected from the tissue.
  • One embodiment of the detection method (test method, diagnostic method) of the present invention using the CTL detection agent of the present invention is carried out by detecting and measuring the amount of peptide-specific CTL of the present invention in a biological sample.
  • a tetramer (HLA tetramer) of a complex between a fluorescently labeled HLA antigen and the peptide of the present invention is prepared according to the method described in the literature (Science, 274: p. 94, 1996, which is incorporated herein by reference), and the antigen peptide-specific CTL in peripheral blood lymphocytes of a patient suspected of having cancer is quantified by a flow cytometer using this.
  • the prediction, determination, judgment, or diagnosis of the presence or absence of a tumor can be performed, for example, by measuring the amount of CTL specific to the peptide of the present invention or the amount of cells presenting the peptide of the present invention in the blood of a subject or a test tissue suspected of having a tumor, and in some cases, the hERV gene expression level, the peptide level of the present invention, or the CTL level in a normal corresponding tissue is used as a reference value, and the reference value is compared with the above-mentioned level in a sample obtained from the subject, and the difference between the two is determined.
  • the comparison of the levels between the test tissue of the subject and the corresponding normal tissue can be carried out by performing measurements of the biological sample of the subject and the biological sample of the normal subject in parallel. If parallel measurements are not performed, the average or statistical median of the amount of the peptide-specific CTL of the present invention or the amount of the cells presenting the peptide of the present invention obtained by measuring multiple (at least two, preferably three or more, more preferably five or more) normal tissues under uniform measurement conditions can be used for comparison as the value of the normal subject, i.e., the reference value.
  • Whether or not a subject is suffering from cancer can be determined, for example, by using as an indicator the amount of CTLs specific to the peptide of the present invention in the tissue of the subject, or the amount of cells presenting the peptide of the present invention, which is, for example, at least two-fold, preferably at least three-fold, higher than the levels in normal subjects.
  • the amount of CTLs specific to the peptide of the present invention in the tissue of the subject is, for example, at least two times, preferably at least three times, higher than the level in a normal subject, and this can be used as an indicator to determine whether a treatment using the peptide or polynucleotide of the present invention is effective.
  • the present invention also relates to a method for preventing and/or treating cancer in a subject, the method comprising the step of administering to a subject in need thereof an effective amount of an active ingredient selected from the group consisting of the peptide, polynucleotide, CTL, antigen-presenting cell, TCR-like antibody, artificial CTL, and genetically modified T cell of the present invention.
  • the "subject" in the present invention may be any individual organism that can be affected by cancer, but is preferably a human or non-human mammalian individual (e.g., rodents such as mice, rats, guinea pigs, and hamsters, primates such as chimpanzees, even-toed ungulates such as cows, goats, and sheep, odd-toed ungulates such as horses, rabbits, dogs, cats, etc.), and more preferably a human individual.
  • the subject may be healthy or may be affected by some disease, but when prevention and/or treatment of cancer is intended, the subject typically means a subject that is affected by or at risk of developing cancer.
  • the subject is HLA-A24 positive. In one embodiment of the present invention, the subject is affected by or at risk of developing hERV-positive cancer. In one embodiment of the present invention, the subject is HLA-A24 positive and is affected by or at risk of developing hERV-positive cancer.
  • the peptides, polynucleotides, CTLs, antigen-presenting cells, TCR-like antibodies, artificial CTLs, and genetically modified T cells of the present invention used in the preventive/therapeutic methods of the present invention include any of those described herein.
  • the effective amount in the present invention is, for example, an amount that reduces the symptoms of cancer or delays or stops its progression, and preferably an amount that suppresses or cures cancer. In addition, an amount that does not cause adverse effects that exceed the benefits of administration is preferable. Such an amount can be appropriately determined by in vitro tests using cultured cells or tests in model animals such as mice and rats, and such test methods are well known to those skilled in the art.
  • the specific dose of the active ingredient can be determined taking into consideration various conditions related to the subject in need thereof, such as the severity of symptoms, the general health condition of the subject, age, weight, sex of the subject, diet, timing and frequency of administration, concomitant medications, responsiveness to treatment, dosage form, and compliance with treatment.
  • the peptide of the present invention is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 10 mg, and it is preferable to administer this once every few days to several months.
  • the polynucleotide of the present invention it is usually 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg, and it is preferable to administer this once every few days to several months.
  • the TCR-like antibody of the present invention it is usually 0.0001 mg to 2000 mg, preferably 0.001 mg to 2000 mg, and it is preferable to administer this once every 1 to 4 weeks.
  • the genetically modified T cell or artificial CTL of the present invention it is usually 1 x 10 4 to 1 x 10 8 , preferably 1 x 10 5 to 1 x 10 7 , and it is preferable to administer this once every 1 day to 4 weeks.
  • any known appropriate administration method can be used, such as intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, etc.
  • an ex vivo method can also be used in which certain cells are collected from a human, CTLs or antigen-presenting cells are induced ex vivo using the peptide or polynucleotide of the present invention, and then these cells are returned to the body.
  • One embodiment of the preventive/therapeutic method of the present invention further comprises a step of selecting an HLA-A24-positive subject as a subject for prevention/treatment before the administration step.
  • This embodiment of the present invention may further comprise a step of determining the HLA type of the subject before the selection step. The determination of the HLA type of the subject can be performed by any known method.
  • one embodiment of the preventive/therapeutic method of the present invention further comprises a step of selecting a subject having hERV-positive cancer as a subject for prevention/treatment before the administration step.
  • This embodiment of the present invention may further comprise a step of detecting hERV-positive cancer in the subject before the selection step.
  • the tumor detection method described in ⁇ 11> above can be used to detect hERV-positive cancer in the subject.
  • One embodiment of the preventive/therapeutic method of the present invention further comprises a step of selecting a subject who is HLA-A24-positive and has hERV-positive cancer as a subject for prevention/treatment before the administration step.
  • This aspect of the invention may further include, prior to the selecting step, determining the subject's HLA type and detecting hERV-positive cancer in the subject.
  • the expression level of the hERV expression product in the detection subject is considered to be positively correlated with the amount of tumor cells in the detection subject. Therefore, by comparing the expression level of the hERV expression product before and after administration of a candidate compound for a cancer therapeutic drug to the detection subject, it is possible to determine whether the administered candidate compound is useful as a cancer therapeutic drug targeting tumor cells.
  • the screening method of the present invention comprises the following steps (I), (II) and optionally (III): (I) measuring the detected amount A of the expression product of the hERV gene in a subject before administering a candidate compound for a cancer therapeutic agent to the subject; (II) administering the candidate compound to the subject cell population and then measuring a detected amount B of the expression product of the gene in the subject; and (III) comparing the detected amounts A and B, and determining that the candidate compound is a candidate cancer therapeutic agent that targets cancer cells if the detected amount A is significantly greater than B.
  • a specific embodiment of the screening method of the present invention comprises the above steps (I) to (III), wherein the steps of measuring the detection amount in steps (I) and (II) comprise steps (d) and (e) in the above detection (test, diagnosis) method, respectively.
  • the present invention relates to a gene expression inhibitor that suppresses the expression of the hERV gene.
  • the method for selectively suppressing the expression of a specific gene in a cell is not particularly limited, and examples thereof include the antisense RNA method, the RNA interference (RNAi) method, the CRISPR-Cas method, the ZFN method, the TALEN method, etc.
  • the antisense RNA method and the RNAi method are preferred, and the RNAi method is more preferred.
  • the gene expression inhibitor is an antisense oligonucleotide against the hERV gene.
  • an "antisense oligonucleotide" against a certain gene means an oligonucleotide that can suppress the expression of the gene by hybridizing to the mRNA, which is the expression product of the gene, and may be any nucleic acid such as DNA or RNA.
  • Such an oligonucleotide is typically an oligonucleotide having a sequence complementary to a portion of the sequence of the mRNA of the gene.
  • complementary means that a certain nucleic acid can form hydrogen bonds with another nucleic acid sequence
  • a specific "sequence complementary to (a portion of) a sequence” means a sequence that has complementarity to the extent that it can hybridize with a nucleotide having the sequence in the intracellular environment. Therefore, it is not necessary that the entire sequence is complementary (i.e., completely complementary).
  • the antisense oligonucleotides of the present invention typically have a length of about 15 to 30 nucleotides.
  • they may be modified as known in the art for the purpose of improving in vivo stability and expression-suppressing activity, reducing off-target effects, etc.
  • the gene expression inhibitor is an siRNA for the hERV gene.
  • siRNA for a gene means a double-stranded RNA capable of inhibiting the expression of the gene, the double-stranded RNA having a sense region and an antisense region, the antisense region being complementary to the sequence of the mRNA of a specific gene, and the sense region being complementary to the sequence of the antisense region.
  • Each of the sense region and antisense region of the siRNA of the present invention has a length of about 15 to 30 nucleotides, preferably about 19 to 27 nucleotides.
  • the sense region and the antisense region may each form a double-stranded structure by two strands, the sense strand and the antisense strand.
  • the sense region and the antisense region may be linked to form one nucleotide strand, in which case the single-stranded RNA is folded into a hairpin shape, and the sense region and the antisense region form a double-stranded structure.
  • siRNA of the present invention can be appropriately modified or altered by these known methods to improve the function of siRNA.
  • the above polynucleotides can be easily synthesized by methods known in the art, for example, by using a commercially available DNA synthesizer.
  • the above-mentioned pharmaceutical composition containing the polynucleotide of this embodiment can be used as a cancer prevention and/or treatment agent.
  • the present invention also relates to a method for preventing and/or treating cancer, which comprises suppressing the expression of the above-mentioned hERV gene.
  • This method can be carried out in accordance with the method described in ⁇ 12> above, except that the administered active ingredient is a gene expression inhibitor that suppresses the expression of the hERV gene, preferably an antisense oligonucleotide or siRNA that suppresses the expression of the hERV gene.
  • the above method can be said to be a method for preventing and/or treating cancer, which includes a step of administering an effective amount of an inhibitor of hERV gene expression to a subject in need thereof.
  • the subject may be healthy or suffering from any disease, but when cancer prevention and/or treatment is intended, the subject typically means a subject suffering from or at risk of suffering from cancer.
  • the subject is suffering from or at risk of suffering from hERV-positive cancer.
  • one embodiment of the prevention/treatment method of the present invention may further include a step of selecting a subject having hERV-positive cancer as a subject for prevention/treatment prior to the administration step.
  • the method for detecting hERV-positive cancer in a subject described above in ⁇ 11> may be used.
  • the effective amount is, for example, an amount that reduces the symptoms of cancer or delays or stops its progression, and is preferably an amount that inhibits or cures cancer.
  • an amount that does not cause adverse effects that exceed the benefits of administration is preferable.
  • Such an amount can be appropriately determined by in vitro tests using cultured cells or tests in model animals such as mice and rats, and such test methods are well known to those skilled in the art.
  • the specific dose of the active ingredient can be determined taking into consideration various conditions related to the subject in need thereof, such as the severity of symptoms, the subject's general health condition, age, weight, sex, diet, timing and frequency of administration, concomitant medications, responsiveness to treatment, dosage form, and compliance with treatment.
  • RNA-seq(RNA-sequencing) Total RNA was isolated from tumor or normal kidney tissues using the Allprep DNA/RNA/Protein Kit (Qiagen) or TRIzol Reagent (Invitrogen) with verified amounts of RNA integrity number (RIN) > 7. Selected libraries were prepared and sequenced by Macrogen (Japan) with 200M 100-bp paired-end reads per sample (18) . Gene or transcript abundance was calculated as TPM. Marker genes for immune cells were selected and grouped as previously described (19) . hERV gene expression was calculated using hervQuant (12) .
  • HLA ligands were eluted with 0.2% TFA and desalted using a Sep-Pak tC18 (Waters) with 28% ACN in 0.1% TFA and a ZipTip U-C18 (Millipore) with 50% ACN in 1% TFA. Samples were dried by vacuum centrifugation and resuspended in 5% ACN in 0.1% TFA for LC-MS/MS analysis.
  • C7709A2 hybridoma (kindly provided by Dr. P.G.
  • a custom database for proteogenomic identification MS searches of hERV-derived antigens was constructed using Python scripts.
  • the database contained three sets of sequences: (a) GENCODE protein-coding transcript translated sequences (31) ; (b) protein sequences altered with missense or frameshift mutations found in samples starting 30 amino acids upstream of the mutated residue and ending at a residue 30 amino acids downstream (missense) or at a stop codon (frameshift); (c) hypothetical protein sequences derived from hERV, where the possible open reading frames found in hERV starting with ATG and ending at a stop codon were translated into three frames. Only protein sequences with gene expression (TPM>0) were included in the database. hERV expression was calculated using hervQuant (12) .
  • MS/MS data were searched against a custom database using Sequest HT and the Percolator algorithm on the Proteome Discoverer 2.3 platform (Thermo). Precursor and fragment ion tolerances were set to 10 ppm and 0.02 Da, respectively. Methionine oxidation (+15.995 Da) was selected as the dynamic modification. No specific enzyme was selected for the search. Concatenated target decoy selection was validated based on q-values, and a false positive rate (FDR) of 0.01 was used in the Percolator node as the peptide detection threshold. Peptides of 8-12 mers were counted as natural HLA-A * 24:02 ligands.
  • FDR false positive rate
  • TILs Tumor infiltrating lymphocytes
  • AIM-V medium was supplemented with 1% penicillin-streptomycin, 1% GlutaMAX (Gibco), 10 mM HEPES, 1 mM sodium pyruvate, 55 ⁇ M 2-mercaptoethanol, and 10% human AB serum (Biowest).
  • the expanded cells were cultured for 2 weeks in AIM-V supplemented with 6,000 U/mL IL-2, 30 ng/mL anti-CD3 (OKT3, BioLegend 317302), and 2.5 ⁇ g/mL amphotericin B with irradiated (100 Gy) HD-derived PBMS.
  • the expanded cells were cryopreserved until use as TILs.
  • IFN ⁇ ELISpot PBMCs were isolated from peripheral blood of three HDs using Lymphoprep (CosmoBio) according to the manufacturer's instructions. Cells were incubated with 1 ⁇ M LF9 peptide or GK12 peptide (negative control) on day 0. and stimulated on day 7, and 50 U/mL rhIL-2 (Peprotech) was added on day 1. On day 14, T2 cells expressing HLA-A * 24:02 (T2-A24, K. Kuzushima et al. (gift from Dr. Aichi Cancer Center Research Institute, Nagoya) was incubated with 1 ⁇ M LF9 synthetic peptide or alone for 2 hours at 25° C.
  • HD PBMS was mixed with T2-A24 cells at a ratio of 10:1.
  • the mixture was mixed and cultured on a human IFN ⁇ ELISPOT plate (BD) at 37° C. for 24 hours.
  • the contents of the plates were reacted with biotinylated anti-human IFN ⁇ for 2 hours at 25° C., followed by ELISPOT streptavidin-HRP for 1 hour at 25° C. Positive spots were identified using the ELISPOT AEC Substrate Set (BD).
  • Synthetic peptides (LYDTVTHTF, LF9; GYISPYFINTSK, GK12) of >80% purity were purchased (Sigma and CosmoBio).
  • Flow cytometry TILs or stimulated HD PBMCs were prestained with human FcR blocking reagent (Clear Back, MBL) and stained with LF9- or HIV env584-592 -HLA-A24 tetramers conjugated with PE or FITC for 24 min at 4° C., followed by staining with anti-CD8-PC5 (Beckman Coulter) for 20 min at 4° C. Cells were also stained with anti-CD8-APC (BioLegend), anti-CD4-PE-Cy7 (BioLegend), and anti-CD3-PE (BioLegend) for 20 min at 4° C. Stained cells were analyzed using a FACSCantoII (BD) in conjunction with a FACSDiva (BD). LF9- or HIV env584-592 -HLA-A24 tetramers were purchased from MBL.
  • RNA extraction was performed as previously described (20) .
  • RNA samples were treated with RNase-Free DNase Set (Qiagen).
  • cDNA was extracted from cancer or normal tissue by reverse transcription using SuperScriptIII (Invitrogen). The cDNA was synthesized from 0.25 ⁇ g of total RNA isolated from kidney tissue.
  • a panel of cDNAs from human normal tissues was purchased from ClonTech.
  • Example 1 Tumor Microenvironment and Immune Profiling of Renal Cell Carcinoma (RCC) RNA sequencing was performed using tumor tissues collected from three patients with renal clear cell carcinoma who underwent nephrectomy. A set of immune cell-related genes was classified and their expression levels were compared across samples ( Figure 1A) (19) . This analysis suggested that T cells and macrophages were recruited in the RCC17 tumor microenvironment (TME). Notably, these markers revealed the activation of CD8 + cytotoxic T cells. Gene expression was further compared between RCC17 tumor and normal tissue lesions ( Figure 1B).
  • Example 2 Landscape of peptides presented by HLA class I in RCC tissues A proteogenomic approach using mass spectrometry (MS) was used to explore the identity of antigens recognized by CD8 + tumor-infiltrating T lymphocytes (TILs) (Figure 2A) (14,15) .
  • This approach allows for the direct and comprehensive analysis of the HLA-presented immune peptidome, including genetic mutation-derived neoantigens, in tissue samples from epithelial solid tumors and non-epithelial tumors or lymph nodes (18,21) .
  • Peptide-HLA-A24 complexes were immunoprecipitated using specific antibodies, and eluted peptides were analyzed using MS. After completing the MS database search, stringency was ensured by selecting only sequences with false discovery rates (FDR) of 0.01 and lengths between 8 and 12 amino acids. A total of 2,294 non-redundant reference peptides were identified by analysis of RCC17 tumor and normal tissues.
  • RNA-seq data were analyzed using hervQuant to select hERVs expressed in RCC17 from 3,173 reported hREV genes (12, 23) .
  • a custom reference database was prepared containing hypothetical protein sequences originating from potential open reading frames (ORFs) from hERV. The addition of this database allowed the identification of eight additional peptides from tumor and normal samples (Table 1) (corresponding to SEQ ID NOs: 3-10, from top to bottom).
  • Example 3 Discovery of tumor-associated hERV peptides Among the eight hERV-derived HLA ligands identified in RCC17, four and three peptides were detected exclusively in normal and tumor tissues, respectively, and one peptide was shared by these samples ( Figure 3A and Table 1). We focused on the four hERV peptides found exclusively in tumors to assess their potential as tumor-associated antigens. Differential gene expression analysis between tumor and normal tissues revealed that herv3895, one of the four hERV encoding peptides, was increased 7.7-fold in tumors (Figure 3B). herv3895 expression was also high in RCC19 and RCC21 tumor tissues ( Figure 3C).
  • RT-qPCR Quantitative reverse transcription PCR
  • LF9 was encoded at the 3' end of a latent ORF, which was neither the first ORF nor long enough to encode a consensus protein sequence (Figure 3E).
  • the MS/MS signal of LF9 detected in RCC17 tumor tissues was verified using a synthetic peptide ( Figure 3F).
  • Example 4 Identification of tumor-associated immunogenic hERV antigens
  • RCC17 tumor tissue was minced and patient TILs were expanded in vitro for 4 weeks. Expanded TILs contained ⁇ 30.2% CD8 + and 68.6% CD4 + T cells (Figure 4A). Although in vitro expansion often introduces a bias in the TCR repertoire in accordance with variations in T cell proliferation (24) , analysis using LF9-HLA-A24 tetramers revealed a fraction of CD8 + T cells that specifically recognized LF9 ( Figure 4B). Infiltration of LF9-specific CD8 + T cells into the TME suggested the immunogenicity of LF9 to induce a spontaneous host immune response in vivo, supporting its role as a tumor-associated antigen in the clinical setting.
  • LF9 The immunogenicity of LF9 appears to be due to its biased HLA presentation to tumor cells (Table 1). Overexpression of the hERV3985 gene in tumors is one of the possible explanations for tumor-specific HLA presentation. However, it remains unclear whether LF9 is the immunodominant antigen responsible for CD8 + T infiltration in RCC17 tumors. The limited number of LF9-reactive TILs precluded sequencing their TCR and estimating their frequency in the TME. Furthermore, since the HLA-A24 immune peptidome has been focused and explored in RCC17, possible neoantigen presentation by HLA class I alleles other than HLA-A24 cannot be excluded.
  • hERV antigens may be useful as tumor antigens shared among HLA-matched patients. Recently, such shared hERV antigens were identified in patients with breast cancer (28) . In our study, the expression level of hERV3895 encoding LF9 was not only high in RCC17 but also in RCC19 and RCC21 tumors ( Figure 3C).
  • LF9 is a tumor antigen that is shared among patients and potentially serves as a ready-made target for antigen-recognition immunotherapy, such as vaccination or genetically engineered T cell therapy.
  • Retroviral-K Envelope Is a Novel Tumor Antigen and Prognostic Indicator of Renal Cell Carcinoma. Front Oncol. 2021;11(657187. 26. Cherkasova E, Scrivani C, Doh S, Weisman Q, Takahashi Y, Harashima N, Yokoyama H, Srinivasan R, Linehan WM, Lerman MI, et al. Detection of an Immunogenic HERV-E Envelope with Selective Expression in Clear Cell Kidney Cancer.

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Abstract

Le but de la présente invention est de fournir un agent de détection pour détecter spécifiquement une cellule tumorale, un peptide d'antigène tumoral présenté de manière spécifique d'une cellule tumorale, et une composition pharmaceutique ou similaire contenant le peptide d'antigène tumoral en tant que principe actif et utile pour la prévention et/ou le traitement du cancer.　Le problème a été résolu par un peptide d'antigène tumoral qui comprend de 8 à 14 résidus d'acides aminés contigus situés dans une séquence d'acides aminés pour une protéine codée par un gène de rétrovirus endogène humain (hERV) et qui a une activité de liaison à HLA, ou un corps substitué par un motif du peptide d'antigène tumoral.
PCT/JP2024/022321 2023-06-19 2024-06-19 Peptide d'antigène tumoral codé par un gène de rétrovirus endogène humain Pending WO2024262558A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013165722A (ja) * 2000-12-07 2013-08-29 Novartis Vaccines & Diagnostics Inc 前立腺癌においてアップレギュレートされた内因性レトロウイルス
WO2021198449A2 (fr) * 2020-04-02 2021-10-07 Istituto Nazionale Tumori Irccs - Fondazione G. Pascale Antigènes tumoraux pour l'immunothérapie du cancer du foie
WO2022152880A1 (fr) * 2021-01-15 2022-07-21 Immatics Biotechnologies Gmbh Peptides presentés par les hla destinés à être utilisés en immunothérapie contre différents types de cancers

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013165722A (ja) * 2000-12-07 2013-08-29 Novartis Vaccines & Diagnostics Inc 前立腺癌においてアップレギュレートされた内因性レトロウイルス
WO2021198449A2 (fr) * 2020-04-02 2021-10-07 Istituto Nazionale Tumori Irccs - Fondazione G. Pascale Antigènes tumoraux pour l'immunothérapie du cancer du foie
WO2022152880A1 (fr) * 2021-01-15 2022-07-21 Immatics Biotechnologies Gmbh Peptides presentés par les hla destinés à être utilisés en immunothérapie contre différents types de cancers

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