WO2016203577A1 - Épitope peptidique de cellules t cytotoxiques et utilisation correspondante - Google Patents

Épitope peptidique de cellules t cytotoxiques et utilisation correspondante Download PDF

Info

Publication number
WO2016203577A1
WO2016203577A1 PCT/JP2015/067469 JP2015067469W WO2016203577A1 WO 2016203577 A1 WO2016203577 A1 WO 2016203577A1 JP 2015067469 W JP2015067469 W JP 2015067469W WO 2016203577 A1 WO2016203577 A1 WO 2016203577A1
Authority
WO
WIPO (PCT)
Prior art keywords
ctl
ebv
peptide
epitope peptide
specific
Prior art date
Application number
PCT/JP2015/067469
Other languages
English (en)
Japanese (ja)
Inventor
棟梁 李
弘紀 大鷹
一絵 中野
真悟 田路
Original Assignee
株式会社医学生物学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社医学生物学研究所 filed Critical 株式会社医学生物学研究所
Priority to JP2017524207A priority Critical patent/JPWO2016203577A1/ja
Priority to CN201580080723.XA priority patent/CN107709351A/zh
Priority to PCT/JP2015/067469 priority patent/WO2016203577A1/fr
Priority to TW105119067A priority patent/TW201708248A/zh
Publication of WO2016203577A1 publication Critical patent/WO2016203577A1/fr

Links

Images

Classifications

    • 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
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a cytotoxic T cell (cytotoxic T T lymphocyte, hereinafter referred to as CTL) epitope peptide specific to Epstein-Barr virus (hereinafter referred to as EBV), and EBV using the peptide.
  • CTL cytotoxic T T lymphocyte
  • EBV Epstein-Barr virus
  • the present invention relates to a vaccine for treating or preventing infection and virus-positive cancer, a passive immunotherapeutic agent for EBV, and a method for quantifying EBV-specific CTL.
  • the present invention relates to a peptide capable of inducing CTL targeting cancer cells.
  • this invention relates to the cancer vaccine and anticancer agent containing the said peptide.
  • the present invention further relates to the use of the peptide for inducing CTL targeting cancer cells, the obtained CTL and an anticancer agent comprising the CTL.
  • Epstein and Barr discovered a new herpes virus from cultured cells derived from Burkitt lymphoma tissue and named it Epstein-Barr virus (EBV) (Non-patent Document 1).
  • EBV is classified into HHV-4, which is one of eight types of human herpesviruses (HHV), and is a virus that is widely latently infected worldwide (Non-patent Document 2).
  • HHV-4 human herpesviruses
  • Non-patent Document 2 a virus that is widely latently infected worldwide
  • infection occurs in the oral and pharyngeal mucosa via saliva in childhood, and the virus produced in the oral and pharyngeal mucosal epithelial cells further infects B cells that pass between the epithelia and spreads throughout the body.
  • Infected B cells are injured and killed mainly by the immune surveillance mechanism with cytotoxic T cells (CTL), but some become latent infections that do not produce viruses.
  • CTL cytotoxic T cells
  • EBV virus-derived DNA fragments were found in nasopharyngeal carcinoma tissues in 1970, EBV has been called the first tumor virus among viruses that infect humans (Non-Patent Documents 3 and 4).
  • typical examples of tumor viruses include hepatitis C virus (HCV) that causes liver cancer, human papilloma virus (HPV) that causes cervical cancer, and humans that cause adult T-cell leukemia.
  • HCV hepatitis C virus
  • HPV human papilloma virus
  • HTLV-1 T cell leukemia virus
  • EBV mainly infects B cells, one of the human lymphocyte components, but it also infects epithelial cells, T cells, NK cells, etc. in addition to B cells, and its diverse cell tropism causes various tumor development Is thought to be involved.
  • cancers that are thought to be caused by EBV infection include malignant tumors such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), NK / T cell lymphoma, nasopharyngeal cancer (NPC), and gastric cancer (MK). It is done.
  • BL Burkitt lymphoma
  • HL Hodgkin lymphoma
  • NPC nasopharyngeal cancer
  • MK gastric cancer
  • LPD lymphoproliferative disease
  • IM infectious mononucleosis
  • PTLD post-transplant lymphoproliferative disease
  • EBV-derived small RNA EBV encoded small RNA, EBER
  • EBER EBV encoded small RNA
  • EBV lymphoproliferative disease
  • PTLD post-transplant lymphoproliferative disease
  • immune cells that transfuse immunocompetent cells that can eliminate the causative virus-infected cells into the patient's body Therapy was performed and the therapeutic effect was proved
  • Non-patent Documents 8, 9, and 10 The effectiveness of immune cell therapy has also been reported for EBV-related malignant tumors such as Hodgkin lymphoma and nasopharyngeal cancer.
  • Immune cell therapy is considered to be a powerful treatment for malignant tumors and immunodeficiency diseases that are refractory to conventional surgical procedures, radiation therapy, and chemotherapy.
  • Immune cell therapy is a treatment method that activates the immune power of patients and specifically attacks and eliminates target cancer cells or virus-infected cells. is there.
  • Cytotoxic T cells (CTL) play a central role in specifically attacking these target cells.
  • CTL Cytotoxic T cells
  • TCR T cell receptor
  • TCR does not directly recognize cancer antigen molecules or virus particles themselves, but HLA (human leukocyte type antigen) expressed on the membrane surface of target cells and, in the case of tumor cells, cancer antigen-derived or viral infection
  • HLA human leukocyte type antigen
  • CTL recognizes a target cell and exerts a killing effect by binding to a complex with a peptide (epitope peptide) consisting of 8 to 10 amino acids derived from a virus.
  • HLA is broadly classified into class I and class II. Complexes of HLA class I and peptides are recognized by TCR expressed on CD8 + T cells, and complexes of HLA class II and peptides are TCR expressed on CD4 + T cells. And an immune response is elicited.
  • HLA class I is further divided into a classical classification called HLA-A, B, C and a non-classical classification called HLA-E, F, G.
  • HLA compatibility between donors and recipients in transplantation therapy is important for HLA-A, B and HLA-DRB class 6 HLA-DRB classification, especially for transplants between unrelated individuals. It is considered as a risk factor.
  • IMGT HLA database http://www.imgt.org/
  • 2,041 types of HLA-A, 2,688 types of HLA-B, and 1,677 types of proteins are registered for HLA-C.
  • HLA-A11 is over 50% of white people, and about 20 Japanese people % Holds.
  • the EBV-derived LMP2 and EBNA1-specific CTL epitope peptides identified by the inventors specifically bind to HLA-A11, and EBV-infected cells are killed and eliminated by CTLs that recognize them.
  • the ownership rate of HLA-A11 is about 10% in the Japanese, but in Southeast Asia, it has a wide population distribution as the third largest allele.
  • HLA-A24, HLA-A2 and HLA-A11 in Southeast Asia are 32.1%, 24.5% and 23.7%, respectively (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/ ihwg.cgi).
  • NPC nasopharyngeal Carcinoma
  • GLOBOCAN2012 GLOBOCAN2012
  • HLA type has been reported as one of the onset risks of NPC (Non-patent Document 17).
  • the frequency of HLA-A which is the highest in the results of statistical analysis of HLA-A frequency in NPC patients, is 50% for A11 holders, 50% for A2 holders, and A24 holders. 30% and B40 holders were reported to be 32% (Non-patent Document 15).
  • HLA-A11 has been reported to be closely related to the onset of NPC (Non-patent literature). 19). In this way, NPC has a high incidence of HLA-A11 carriers, and it is probable that it is profound to provide a treatment method using EBV LMP2 and EBV EBNA1-specific CTL epitopes exhibiting HLA-A11 restriction according to the present invention.
  • NPC is a refractory malignant tumor
  • CTL epitope peptides used in these clinical trials are mainly HLA-A2 and HLA-A24 restricted peptides, and there are almost no clinical trial reports of HLA-A11 restricted CTL epitope peptides.
  • the reason for this is that almost no attempt has been made worldwide to identify HLA-A11-restricted CTL epitope peptides and it is very difficult to identify EBNA1-derived CTL epitopes expressed in NPCs. Can be mentioned.
  • the identification method described later was used to intensively investigate the identification of CTL epitope peptides that are restricted to EBV-related malignant tumors, particularly HLA-A11, which has the highest prevalence in NPC patients.
  • EBV has about 170kbp double-stranded DNA encoding about 85 gene products, but the expression of LMP1, LMP2, and EBNA1, which are EBV-derived proteins, has been reported in NPC cancer affected areas. While LMP1 can be detected only in some patients, LMP2 and EBNA1 are constitutively expressed in NPC (Non-patent Documents 20 and 21), and these two proteins are used to identify cytotoxic T cell epitope peptides. Is an attractive target for. However, in previous reports, it has been believed that the immune response of CTL is preferentially directed to LMP2, and that EBNA1 is not recognized by CTL (Non-patent Documents 22, 23, 24, 25).
  • EBNA1 is composed of 641 amino acids in total length, but there is a glycine-alanine repetitive sequence (GAr) consisting of about 200 amino acids in the central region (101st to 324th amino acids).
  • Gr glycine-alanine repetitive sequence
  • sequences corresponding to specific CTL epitope peptides may differ between EBV strains, for example, B95.8 strain and GD1 strain, which may affect CTL induction ability and detection ability with MHC-tetramer reagent.
  • MHC major histocompatibility complex
  • HLA major histocompatibility complex
  • MHC class II (hereinafter referred to as MHC-II) is expressed on the cell membrane surface of antigen-presenting cells such as dendritic cells, and presents non-self peptides to CD4 + T cells to induce cytokine secretion and antibody production To do.
  • MHC-I-CTL MHC-II-CD4 + T cells
  • Non-patent Document 31 This is the first example that shows that MHC-I specifically induces cytotoxic activity by presenting its own antigenic peptide derived from cancer cells to CTL.
  • CTL can also react with self-antigens, and it has been shown that cancer can be treated by using its function. Since then, identification of proteins specifically expressed in cancer (hereinafter referred to as cancer antigens) and peptide fragments derived from the proteins has been actively promoted. In parallel with these discoveries, development of cancer immunotherapy has been actively promoted. Cancer immunotherapy is a method for treating cancer by proliferating immunocytes such as CTLs that kill and eliminate cancer cells in vivo and in vivo. There are several possible methods for cancer immunotherapy.
  • 1 to 3 are non-specific cancer immunotherapy, which is not necessarily an immune response specific to cancer antigens.
  • a surgical procedure such as excision of a cancer affected part from a cancer patient becomes necessary.
  • Cancer peptide vaccine therapy in which a patient is inoculated with a cancer antigen-specific CTL epitope peptide and an immunostimulant.
  • Peptide pulse dendritic cell therapy in which a cancer antigen-specific CTL epitope peptide is pulsed to a dendritic cell isolated and cultured from peripheral blood of a patient and then inoculated to the patient.
  • Combination therapy of CTL transfer therapy 7.4 and 6 that induces specific CTL by stimulating culture of lymphocytes isolated from patient's peripheral blood using cancer antigen-specific CTL epitope peptide and returns this to the patient's body.
  • the patient is inoculated with a cancer antigen-specific CTL epitope peptide and an immunostimulant.
  • This method induces specific CTLs by stimulating the lymphocytes isolated from the patient's peripheral blood with a cancer antigen-specific CTL epitope peptide and returns it to the patient's body. 8).
  • Artificial CTL transfer therapy by extracting T cell receptor (TCR) gene from cancer antigen-specific CTL and introducing it into lymphocytes isolated and cultured from the patient's peripheral blood to produce artificial CTL and return it to the patient's body .
  • TCR T cell receptor
  • CTL monitoring Cancer immunotherapy is a treatment method that recognizes cancer in vivo and expects amplification of CTL that attacks, and monitoring of CTL, which is an effective component, is important .
  • Methods for monitoring CTL include intracellular cytokine staining and measurement of cytotoxic activity. These are methods for indirectly detecting CTL.
  • intracellular cytokine staining is a method that measures the production of IFN ⁇ (interferon gamma) and TNF ⁇ (tumor necrosis factor-alpha) in response to peptide stimulation, and may detect reactions other than antigen peptide-specific immune responses. is there.
  • IFN ⁇ interferon gamma
  • TNF ⁇ tumor necrosis factor-alpha
  • the MHC-tetramer reagent is a reagent in which a ternary complex (MHC-monomer) of MHC, ⁇ 2-microglobulin (hereinafter ⁇ 2m) and a peptide fragment is produced in a test tube, and the MHC-monomer is tetramerized.
  • MHC-monomer ternary complex of MHC, ⁇ 2-microglobulin (hereinafter ⁇ 2m) and a peptide fragment
  • MHC-MHC that constitutes a monomer is a diverse molecule. According to the IMGT HLA database (http://hla.alleles.org/nomenclature/stats.html), there are 2077 types of HLA-A and HLA-B. There are 2741 types of proteins registered for HLA-C and 1739 types for HLA-C (as of October 2014). In addition, the characteristics of peptide fragments (8 to 12 amino acid residues in length) to be bound differ depending on the allyl type of MHC. In other words, there are an enormous number of combinations of MHC / peptide complexes, which include only the allyl form of MHC and the types of peptide fragments.
  • CTL recognizes the peptide presented by MHC-I by TCR.
  • Individual TCRs are created by rearrangement of the TCR gene, and the number of TCR repertoires in one individual is said to be as high as 10 18 . That is, both the MHC / peptide complex of the target cell and the TCR on the CTL side have a great variety.
  • Each CTL generally expresses one type of TCR on the cell membrane surface and recognizes and activates only a specific MHC / peptide complex.
  • MHC-tetramer reagent is a reagent using this mechanism.
  • the MHC-tetramer reagent is a reagent that mimics the structure of the MHC / peptide complex on the target cell membrane, whereby only CTL having a specific TCR can be selectively detected.
  • the MHC-tetramer reagent is considered to be an ideal reagent for CTL monitoring.
  • the reason that tetramerization of MHC-monomer is to enhance the binding force with TCR expressed by CTLs so that they can be detected by a device such as a flow cytometer.
  • Mutations in antigen-presenting molecules (HLA and ⁇ 2m) in cancer cells Mutations in HLA and ⁇ 2m in cancer cells are known as the immune escape mechanism of cancer cells.
  • cancer cells no longer present the antigenic peptide, so the CTL cannot recognize and attack the cancer cells.
  • an HLA mutation an LOH type mutation (loss of heterozygosity) in which one side of the same locus is deleted is known.
  • ⁇ 2m mutation is known as frameshift mutation.
  • attempts have been made to compensate for ⁇ 2m expression by introducing the ⁇ 2m gene with an adenoviral vector and to present antigenic peptides to cancer cells.
  • (3) Expression of cancer cell target antigens There are various types of cancer antigens, and which cancer antigen is expressed varies depending on the type of cancer and the individual.
  • cancer antigen derived from the peptide vaccine when the cancer antigen derived from the peptide vaccine is not expressed at all in the cancerous part of the patient, the cancer cells cannot be targeted, so the therapeutic effect of the cancer peptide vaccine therapy cannot be expected.
  • cancer peptide vaccine therapy clinical trials have confirmed whether cancer cells of patients express peptide vaccine-derived antigens by tissue staining using cancer antigen-specific antibodies. .
  • malignant glioblastoma it is difficult to confirm the expression of a cancer antigen in a cancer affected part because it is difficult to obtain a cancer cell sample.
  • CTL activates when it specifically recognizes an antigenic peptide presented by a target cell, and exhibits cytotoxic activity.
  • TGF ⁇ is a suppressive cytokine secreted by cancer cells and inhibits the proliferation and differentiation of CTL and CD4 + T cells.
  • PD-1 and CTLA-4 are molecules on the cell membrane of T cells. When each binds to a ligand expressed by cancer cells, an inhibitory signal is transmitted and CTL is inactivated.
  • HLA type of patient Epitope peptide is basically presented to only one type of HLA. This is called HLA restraint. For this reason, even if cancer immunotherapy is performed on a patient who does not have an HLA type capable of presenting an epitope peptide, no therapeutic effect can be expected because cancer cells do not present the epitope peptide in that patient. That is, the target patient of cancer immunotherapy is limited to the HLA restriction
  • HLA-A * 24: 02 is the most common HLA-A allylate in Japanese, with about 60%. Therefore, in the present invention, HLA-A * 24: 02-restricted novel cancer antigen epitope peptide that can be used for specific cancer immunotherapy, cancer vaccine and anticancer agent using the same, and epitope peptide specific
  • An object of the present invention is to provide a reagent that detects CTL.
  • CKAP4 is a type II transmembrane protein composed of a molecular weight of 63 kDa and a total length of 602 amino acids. From the N-terminal side, there are three regions: a cytoplasmic region composed of 106 amino acids, a transmembrane region composed of 21 amino acids, and an extracellular region composed of 475 amino acids (non-patented) Reference 32). CKAP4 has a function of binding to the endoplasmic reticulum and microtubules in the intercytoplasmic region in the interphase of the cell cycle and fixing the endoplasmic reticulum to the microtubules (Non-patent Document 33).
  • Non-patent Document 34 Before the cell enters mitosis, the cysteine residue, which is the 100th amino acid from the N-terminal side of CKAP4, is reversibly palmitoylated (Non-patent Document 34). By this palmitoylation, localization of CKAP4 Turns into a cell membrane (Non-patent Document 35), and the interaction with microtubules is inhibited. It has also been reported that CKAP4 is phosphorylated during mitosis and loses its ability to bind to microtubules (Non-patent Document 36). On the other hand, it is known that microtubules constitute a spindle that plays a role in correctly dividing a cell by distributing chromosomes during mitosis.
  • CKAP4 is thought to be involved in the control of normal mitosis by dissociating appropriately from microtubules during mitosis.
  • CKAP4 is also known to act as a receptor for surfactant-protein-A (hereinafter referred to as SP-A), which is responsible for the removal of surfactant (pulmonary surfactant) on the surface of alveolar cells (non-patented). Reference 32).
  • SP-A surfactant-protein-A
  • SP-A surfactant-protein-A
  • SP-A surfactant-protein-A
  • SP-A surfactant-protein-A
  • SP-A has the function of removing a surfactant and is responsible for maintaining alveolar cell homeostasis by functioning with the receptor CKAP4.
  • CKAP4 is expressed in normal cells, it is highly expressed in cell lines derived from various cancers such as breast cancer, central nervous system tumor, lung cancer, kidney cancer, and malignant melanoma (http://129.187.44.58 : 7070 / NCI60 / protein / show / 10796).
  • CKAP4 is an overexpressed cancer antigen that is highly expressed in cancer cells (Non-patent Document 38).
  • an overexpressed cancer antigen for example, Her-2 is known, and clinical trials of cancer peptide vaccines using Her-2 derived peptides have been conducted (Non-patent Documents 39 and 40).
  • CKAP4 is considered to be highly expressed in various cancers, but the antigenicity of CKAP4 in cancer cells has not been clarified. That is, whether CKAP4 is a target of the immune system as a cancer antigen, that is, CKAP4-derived peptides are presented on the HLA on the surface of cancer cells, and cancer cells attack by CTLs that specifically recognize this It is unclear as to whether or not it has been done.
  • EPSTEIN MA ACHONG BG, BARR YM.Virus particles in cultured lymphoblasts from burkitt's lymphoma. Lancet. 1964 Mar 28; 1 (7335): 702-3.
  • Ikuta K Satoh Y, Hoshikawa Y, Sairenji T. Detection of Epstein-Barr virus in salivas and throat washings in healthy adults and children. Microbes Infect. 2000 Feb; 2 (2): 115-20.
  • Pagano JS Epstein-Barr virus: the first human tumor virus and its role in cancer. Proc Assoc Am Physicians. 1999 Nov-Dec; 111 (6): 573-80.
  • Vedrenne C Klopfenstein DR, Hauri HP. Phosphorylation controls CLIMP-63-mediated anchoring of the endoplasmic reticulum to microtubules. Mol Biol Cell. 2005; 16: 1928-1937. Li SX, Tang GS, Zhou DX, Pan YF, Tan YX, Zhang J, Zhang B, Ding ZW, Liu LJ, Jiang TY, Hu HP, Dong LW, Wang HY. Prognostic significance membrane associated membrane associated palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma. Cancer. 2014; 120: 1520-1531. Heemskerk B, Kvistborg P, Schumacher TN. The cancer antigenome. EMBO J.
  • the present invention has been made in view of such a situation, and the target is limited to LMP2 and EBNA1 of EBV, and the purpose thereof is LMP2-specific cytotoxic T cell epitope peptide, and EBNA1-specific cytotoxicity.
  • a vaccine for identifying a T cell epitope peptide and treating or preventing EBV-related malignant tumors and immunodeficiencies including NPC using the peptide, a passive immunotherapeutic agent for EBV, and cytotoxic T cells specific to EBV It is to provide a quantitative method.
  • the present inventors have conducted extensive studies on cancer antigenicity, that is, CTL inducing ability of various CKAP4-derived peptides.
  • EBV comprising the steps of obtaining EBV-specific cytotoxic T cells generated by the above steps, and measuring cytokines and / or chemokines and / or cell surface molecules produced by the obtained cytotoxic T cells.
  • a method for quantifying specific cytotoxic T cells [14] A step of mixing the epitope peptide according to any one of [1] to [2], a major histocompatibility antigen complex and ⁇ 2-microglobulin, and the prepared major histocompatibility antigen complex-tetramer and subject-derived A method of quantifying cytotoxic T cells specific for EBV in the peripheral blood, comprising a step of contacting the peripheral blood with [15] A method for inducing cytotoxic T cells specific to EBV, comprising the step of contacting the epitope peptide according to any one of [1] to [2] with an antigen-presenting cell, [16] A kit for inducing cytotoxic T cells comprising the epitope peptide according to any one of [1] to [2] as a constituent element, [17] A method for producing EBV-specific CTL comprising the step of contacting the peptide according to any one of [1] to [2] with peripheral blood mononuclear cells in a medium containing
  • a method for producing a passive immunotherapeutic agent for cancer treatment [33] an antibody specific for a major histocompatibility antigen complex prepared from the epitope peptide of any one of [18] to [19], [A1] Use of the epitope peptide according to any one of [1] to [2] in the manufacture of a medicament for treating or preventing EBV infection or EBV positive cancer, [A2] Use according to [A1], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain, [A3] Use of the nucleic acid or expression vector according to any one of [3] to [4] in the manufacture of a medicament for treating or preventing EBV infection or EBV positive cancer, [A4] Use according to [A3], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.
  • EBV-specific cytotoxic T cells obtained by [A8] Major histocompatibility antigen complex and / or major histocompatibility antigen complex-tetramer prepared from the epitope peptide according to any one of [1] to [2] in the production of a passive immunotherapeutic agent for EBV And peripheral blood lymphocytes are contacted to form a conjugate in which cytotoxic T cells are bound to the major histocompatibility antigen complex and / or major histocompatibility complex-tetramer.
  • cytotoxic T cells obtained separately, [A9] Use of the epitope peptide according to any one of [18] to [19] in the manufacture of a medicament for treating or preventing cancer, [A10] Use of the nucleic acid or expression vector according to any one of [21] to [22] in the manufacture of a medicament for treating or preventing cancer, [A11] Use of an antigen-presenting cell in which the epitope peptide according to any one of [18] to [19] is presented to HLA in the manufacture of a medicament for treating or preventing cancer, [A12] In the production of a passive immunotherapeutic agent for cancer treatment, peripheral blood lymphocytes are produced by the epitope peptide according to any one of [18] to [19] or antigen-presenting cells presenting the epitope peptide on HLA.
  • CKAP4-specific CTL obtained by stimulation [A13] Major histocompatibility complex and / or major histocompatibility antigen prepared from the epitope peptide of any one of [18] to [19] in the production of a passive immunotherapeutic agent for cancer treatment
  • the complex-tetramer is contacted with peripheral blood lymphocytes to form the major histocompatibility antigen complex and / or the conjugate with CTL bound to the major histocompatibility antigen complex-tetramer.
  • a vaccine composition comprising the epitope peptide according to any one of [1] to [2] as an active ingredient for treating or preventing EBV infection or EBV positive cancer
  • the vaccine composition according to [B1] wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain
  • [B3] A vaccine composition comprising the nucleic acid or expression vector according to any one of [3] to [4] as an active ingredient for treating or preventing EBV infection or EBV-positive cancer
  • the vaccine composition according to [B3] wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain, and B95.8 strain
  • [B5] containing, as an active ingredient, an antigen-presenting cell presenting the epitope peptide according to any one of [1] to [
  • a composition comprising cytotoxic T cells obtained by [B9] A vaccine composition comprising the epitope peptide according to any one of [18] to [19] as an active ingredient for cancer treatment or prevention, [B10] A vaccine composition comprising the nucleic acid or expression vector according to any one of [21] to [22] as an active ingredient for cancer treatment or prevention, [B11] A vaccine composition comprising, as an active ingredient, an antigen-presenting cell presenting the epitope peptide according to any one of [18] to [19] on HLA for cancer treatment or prevention, [B12] For treating cancer by passive immunotherapy, the peripheral blood lymphocytes are stimulated by the epitope peptide according to any one of [18] to [19] or antigen-presenting cells presenting the epitope peptide on HLA.
  • a composition comprising CKAP4-specific CTL obtained as an active ingredient, [B13] Major histocompatibility complex and / or major histocompatibility complex prepared from the epitope peptide of any one of [18] to [19] for treating cancer by passive immunotherapy Contacting the tetramer with peripheral blood lymphocytes to form a major histocompatibility antigen complex and / or a major histocompatibility antigen complex-a conjugate in which CTL is bound to the tetramer, and isolated from the conjugate
  • a composition comprising the obtained CTL as an active ingredient, [C1]
  • a method for treating or preventing EBV infection or EBV-positive cancer comprising a step of administering the epitope peptide according to any one of [1] to [2] to an individual in need thereof , [C2] EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain, [C
  • EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain
  • [C5] EBV-infected or EBV virus-positive cancer comprising a step of administering an antigen-presenting cell presenting the epitope peptide of any one of [1] to [2] to HLA to an individual in need thereof
  • a method for treating or preventing [C6] The method according to [C5], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain
  • [C7] EBV-specific cytotoxic T cells obtained by stimulating peripheral blood lymphocytes with the epitope peptide according to any one of [1] to [2] or antigen-presenting cells presenting the epitope peptide on HLA Passive immunotherapy against EBV, comprising the step of administering to an individual in need thereof
  • FIG. 4 is a diagram showing the quantification of the proportion of live IFN ⁇ -producing cells present in CD8-positive cells after confirming EBVEBLMP2-specific CTL induction by the intracellular IFN ⁇ -producing cell quantification method.
  • FIG. 11 is a view showing the detection result of LMP2-specific CTL by the prepared MHC-tetramer reagent (donor ID * 11-11).
  • FIG. 6 is a diagram showing the detection result (1) of EBNA1-specific CTL by the prepared MHC-tetramer reagent.
  • FIG. 6 is a diagram showing the detection result (2) of EBNA1-specific CTL by the prepared MHC-tetramer reagent. It is a figure which shows the confirmation result of EBNA1 specific CTL induction
  • the peptide referred to in the present invention means a molecular chain of linear amino acids having physiological activity and bound to each other by a peptide bond between an ⁇ -amino group and a carboxyl group of adjacent amino acid residues.
  • Peptides are not meant to be of a specific length and can be of various lengths. Further, it may be in an uncharged or salt form, and may be modified by glycosylation, amidation, phosphorylation, carboxylation, phosphorylation or the like in some cases. Further, the epitope peptide of the present invention may be one or several (for example, 1 to 10) unless the physiological peptide and the immune activity are substantially modified and has no harmful activity when administered.
  • Peptides in which insertion, addition, substitution, deletion or the like of amino acids or amino acid analogs have occurred are also included in the present invention.
  • the purpose of changing such amino acids is, for example, 1. Changes to increase affinity with HLA (Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, Restifo NP, Dudley ME, Schwarz SL, Spiess PJ, Wunderlich JR, Parkhurst MR, Kawakami Y, Seipp CA, Einhorn JH, White DE.Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med.
  • the peptide of the present invention can be used in the form of a complex to which saccharides, polyethylene glycol, lipids and the like are added, a derivative with a radioisotope, or a polymer.
  • the amino acid analogs include N-acylated products, O-acylated products, esterified products, acid amidated products, and alkylated products of various amino acids.
  • the N-terminus of the antigen peptide or free amino group May be bound with a formyl group, an acetyl group, a t-butoxycarbonyl (t-Boc) group, and the C-terminus of the antigen peptide and a free carboxyl group include a methyl group, an ethyl group, and a t-butyl group.
  • Group, benzyl group and the like may be bonded.
  • the epitope peptide of the present invention may be subjected to various modifications that can facilitate introduction into the living body.
  • the PT (Protein Transduction) domain is famous as an example of various modifications that can facilitate introduction into the living body.
  • the PT domain of HIV is a peptide composed of the 49th to 57th amino acids (Arg Lys Lys Arg Arg Gln Arg Arg Arg) of the Tat protein. It has been reported that this PT domain can be easily introduced into cells by adding it to the N-terminal and / or C-terminal of the target protein or peptide (Ryu J, Han K, Park J, Choi SY.
  • HSP heat shock prtein
  • HSP90 heat shock prtein
  • gp96 heat shock prtein
  • CD91 is a common receptor for heat shock proteins gp96, hsp70, and calreticulin. Immunity. 2001; 14: 303-313).
  • nucleic acid encoding epitope peptide is important for producing the epitope peptide in the host using genetic recombination techniques. In this case, since the usage frequency (codon usage) of the amino acid codon differs between hosts, it is desirable to change the codon of the amino acid so as to match the codon usage of the host to be produced.
  • Nucleic acids encoding epitope peptides are also important as vaccines and can be transported as bare nucleic acids or using appropriate viral or bacterial vectors (Berzofsky JA, Ahlers JD, Janik J, Morris J, Oh S, Terabe M, Belyakov IM Progress on new vaccine strategies against chronic viral infections.J Clin Invest. 2004; 114: 450-462, Berzofsky JA, Terabe M, Oh S, Belyakov IM, Ahlers JD, Janik JE, Morris JC Progress on new vaccine strategies for the immunotherapy and prevention of cancer. J Clin Invest. 2004; 113: 1515-1525).
  • Suitable bacterial vectors are Salmonella subspecies bacteria.
  • Suitable viral vectors are, for example, retroviral vectors, EBV vectors, vaccinia vectors, Sendai virus vectors, lentiviral vectors.
  • a suitable vaccinia vector is a modified vaccinia ankara vector.
  • the CTL epitope candidate peptide specific for EBV of the present invention consists of 8 to 10 amino acids having a binding motif for the target HLA class I molecule with respect to the amino acid sequences of LMP2 and EBNA1. MHC binding Nucleic Acids Multiple epitope prediction software published on the Internet (Pingping Guan, Irini A. Doytchinova, Christianna Zygouri, and Spotify R. Flower MHCPred: a server for quantitative prediction of peptide? Res., 2003; 31: 3621-3624, Karosiene E, Lundegaard C, Lund O, Nielsen M.
  • NetMHCcons a consensus method for the major histocompatibility complex class I predictions. Immunogenetics. 2012 Mar; 64 (3): 177-86 , Jorgensen KW, Rasmussen M, Buus S, Nielsen M. NetMHCstab-predicting stability of peptide-MHC-I complexes; impacts for cytotoxic T lymphocyte epitope discovery. Immunology. 2014 Jan; 141 (1): 18-26. ) Can be selected.
  • HLA class I molecules mainly include HLA-A, HLA-B, and HLA-C, and the epitope peptides that are displayed by binding to these consist of 8 to 10 amino acids.
  • the second, ninth or tenth amino acid from the N-terminal side of the epitope peptide is the most important amino acid for binding to the HLA class I molecule, and is called an anchor motif. It has been reported that this anchor motif varies depending on the type of each HLA class I molecule. For example, as the peptide that binds to HLA-A2 that is the most studied worldwide, Leu is located at the second position from the N-terminus, and Leu or Val is located at the 9th or 10th position.
  • HLA-A11 Chang CX, Tan AT, Or MY, Toh KY, Lim PY, Chia AS, Froesig TM, Nadua KD, Oh HL, Leong HN, Hadrup SR, Gehring AJ, Tan YJ, Bertoletti A, Grotenbreg GM.
  • peptide library consisting of about 20 amino acid sequences covering the entire target protein among the proteins constituting EBV is synthesized.
  • a library is prepared so that about 10 amino acid sequences out of about 20 amino acids overlap with preceding and subsequent peptides. As a result, the entire protein can be searched comprehensively, and once the library is prepared, the HLA restriction can be comprehensively examined.
  • the CTL epitope candidate peptides represented by SEQ ID NOs: 1 to 35 of the present invention can be prepared by various conventional peptide synthesis methods.
  • organic chemical synthesis methods such as solid phase peptide synthesis methods, or nucleic acids encoding peptides can be prepared and prepared using recombinant DNA technology.
  • combination by a commercially available chemical synthesizer is also possible.
  • the CTL epitope candidate peptide selected by the above-mentioned method does not necessarily become a CTL epitope peptide, and can only become an EBV LMP2-specific CTL epitope peptide and an EBV EBNA1-specific CTL epitope peptide after the following examination.
  • TAP transporter associated with antigen processing
  • HLA class I molecules and ⁇ 2-microglobulin It binds to the complex and is transported to the cell membrane surface.
  • a TAP gene-deficient cell line deficient in this TAP molecule cannot express peptide fragments, which are degradation products of endogenous proteins, on the cell membrane surface.
  • human lymphoblastoid cell line T2 which is a typical TAP gene-deficient cell line, or a cell line (T2-A11) in which HLA-A11 molecule is introduced into T2 is expressed on the cell membrane surface. Very unstable.
  • the HLA molecule when bound to an externally supplied peptide, the HLA molecule is stabilized on the cell membrane surface.
  • the TAP gene-deficient cell line can be used in experiments for verifying the binding properties of HLA molecules and externally supplied peptides. Specifically, mixed culture of TAP gene-deficient cell line and CTL epitope candidate peptide, staining with anti-HLA antibody, and calculating change in expression intensity of HLA molecule by flow cytometry The binding of CTL epitope candidate peptides can be examined.
  • the HLA molecule-peptide complex is stabilized on the cell membrane surface, and when stained with an anti-HLA antibody, the expression of the HLA molecule is enhanced. Is observed.
  • the added CTL epitope candidate peptide does not bind to the HLA molecule, the HLA molecule on the cell membrane surface is unstable, and even when stained with an anti-HLA antibody, enhanced expression of the HLA molecule is not confirmed. Using such a method, it is possible to verify the binding between the HLA molecule and the CTL epitope candidate peptide.
  • the MHC-tetramer reagent is a three-component complex (MHC-monomer) of MHC (HLA in the case of humans), ⁇ 2-microglobulin and peptide fragments in vitro. It is an incorporated reagent.
  • the MHC-tetramer reagent is the only reagent that can selectively detect antigen-specific cytotoxic T cells (CTLs) that are MHC restricted.
  • CTLs cytotoxic T cells
  • MHC-tetramer reagent can not only quantify the number of CTLs by co-staining with anti-CD (cluster of differentiation) antibody, anti-cytokine antibody, etc., but also by analyzing with flow cytometry. It is possible to evaluate every single cell.
  • the first step in MHC-tetramer reagent production begins with folding where the raw materials MHC, ⁇ 2-microglobulin and peptide are mixed in a suitable solution in a test tube.
  • a three-component complex (MHC-monomer) is formed by the association reaction of these three raw materials.
  • MHC-monomer a three-component complex
  • this association reaction proceeds smoothly, and analysis with a gel filtration column makes it possible to detect a complex of three kinds of raw materials (MHC-monomer).
  • MHC-monomer when there is no binding force between MHC and peptide, MHC-monomer is hardly detected. Therefore, it is possible to verify the binding properties and stability of MHC and peptide by analyzing the folding solution over time or by performing heat treatment or the like.
  • Epitope peptide-specific CTL detection (3-1) Epitope peptide determination method Peripheral blood mononuclear cells (PBMC) isolated from a person with a history of EBV infection, or T cells isolated from PBMC Suspend in a suitable medium at a cell concentration of 0.1-2 x 10 6 / mL. To this, 1 ⁇ 10 5 / mL of EBV-infected cells previously separated and cultured from the same person is added, and cultured at 37 ° C. for 7 days in a 5% carbon dioxide (CO 2 ) thermostat.
  • PBMC peripheral blood mononuclear cells
  • EBV-infected cells and interleukin 2 are added, and then CTL are induced by repeated stimulation with EBV-infected cells and IL-2 every week. Whether or not the CTL thus induced is specific for the epitope candidate peptide is determined by MHC-tetramer method, elicitor assay, chromium release assay, intracellular cytokine staining method, etc. (Current Protocols in Immunology Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.
  • Whether or not the CTL thus induced is specific for the epitope candidate peptide is determined by an Elispot assay, a chromium release assay, an intracellular cytokine staining method, or the like. For pooled peptides that showed good results, it is possible to select peptides having CTL inducing ability by repeating the above experiment with one peptide added at a time. The reacted peptides are shortened in order, and finally an epitope peptide consisting of 8 to 10 amino acids is obtained as the epitope peptide of the present invention.
  • EBV LMP2 and EBNA1-specific MHC-monomers and MHC-tetramer reagents [Production of EBV LMP2 and EBNA1-specific MHC-monomers and MHC-tetramer reagents] MHC-monomer and MHC-tetramer reagent using EBV LMP2-specific CTL epitope candidate peptide and EBV EBNA1-specific CTL epitope candidate peptide can be prepared by known methods (US Patent Number 5,635,363, French Application Number FR9911133) .
  • MHC- which is a triple complex of an HLA class I molecule, ⁇ 2-microglobulin and LMP2-specific CTL epitope candidate peptide of the present invention, or EBNA1-specific CTL epitope candidate peptide purified from a recombinant host for protein expression
  • Monomers are formed in the folding solution.
  • a biotin binding site is added to the C-terminus of the recombinant HLA class I molecule in advance, and biotin is added to this site after MHC-monomer formation.
  • a MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4.
  • MHC-tetramer reagent and antibodies against cell surface proteins (CD62L, CCR7, CD45RA, etc.) can be used to examine the differentiation stage of CTL (Seder RA, Ahmed R. Similarities and differences in CD4 + and CD8 + effector and memory T cell generation. Nat Immunol. 2003; 4: 835-842.).
  • it can be used for functional evaluation of CTL by combining with intracellular cytokine staining method.
  • CTL against HCV Hepatitis C virus
  • Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8 + T cells.Blood. 2002; 100: 3690-3697.).
  • a specific CTL epitope peptide is identified and an MHC-tetramer reagent is produced, it becomes possible to quantify and qualify specific CTLs, and to make a great contribution to obtaining diagnostic information.
  • the CTL epitope peptide of the present invention can be used as a peptide vaccine in active immunotherapy. That is, a vaccine comprising the CTL epitope peptide of the present invention is administered to a patient, and EBV LMP2-specific CTL or EBV EBNA1-specific CTL is proliferated in the body to prevent infection and treat infection and EBV positive tumor. Can be useful. Only one type of epitope peptide may be used, or two or more types of peptides may be combined and mixed according to the intended use of the vaccine.
  • Vaccine using antigen-presenting cell The antigen-presenting cell on which the CTL epitope peptide of the present invention is presented can be used as a vaccine in active immunotherapy.
  • Antigen-presenting cells with CTL epitope peptides presented 1.
  • CTL epitope peptide pulse antigen-presenting cells in which antigen-presenting cells and CTL epitope peptides are mixed for 30 minutes to 1 hour in an appropriate culture medium.
  • An antigen-presenting cell means, for example, a dendritic cell, a B cell, a macrophage, a certain type of T cell, etc., and is a cell that expresses on its cell surface an HLA molecule to which the peptide can bind, It means something that has stimulating ability.
  • Artificial antigen-presenting cells with antigen-presenting ability are artificially prepared by immobilizing a ternary complex of HLA molecule, CTL epitope peptide and ⁇ 2-microglobulin on beads such as lipid bilayer membrane, plastic or latex.
  • costimulatory molecules such as CD80, CD83, and CD86 that can stimulate CTLs
  • fix antibodies that act agonistically on CD28, a T cell ligand that binds to costimulatory molecules.
  • Cutting edge predetermined avidity of human CD8 T cells expanded on calibrated MHC / anti -CD28-coated microspheres. J Immunol. 2003; 171: 4974- 4978, Oosten LE, Blokland E, van Halteren AG, Curtsinger J, Mescher MF, Falkenburg JH, Mutis T, Goulmy E. Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes.Blood. 2004; 104: 224 -226).
  • the nucleic acid of the CTL epitope peptide of the present invention can be used for DNA vaccines, recombinant virus vector vaccines and the like in active immunotherapy.
  • it is desirable to change the nucleic acid sequence of the CTL epitope peptide to codon usage suitable for the host producing the recombinant vaccine or the recombinant virus vaccine (Casimiro, DR et al. Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid , Recombinant Vaccinia Virus, and Replication-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene J.
  • a vaccine comprising a CTL epitope peptide of the present invention or an antigen-presenting cell on which a CTL epitope peptide is presented can be prepared using methods known in the art.
  • a vaccine includes an injection or a solid preparation containing the CTL epitope peptide of the present invention as an active ingredient.
  • the CTL epitope peptide can be formulated in a neutral or salt form.
  • pharmaceutically acceptable salts include inorganic salts such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid and tartaric acid. .
  • the antigen-presenting cell on which the CTL epitope peptide of the present invention is presented is a pharmaceutically acceptable excipient compatible with the peptide or the activity of the cell, such as water, saline, dextrose, ethanol, It can be used by mixing with glycerol, DMSO (dimethyl sulphoxide), other adjuvants, or a combination thereof. Furthermore, you may add adjuvants, such as albumin, a wetting agent, and an emulsifier, as needed.
  • the vaccine of the present invention can be administered by parenteral administration or oral administration, but parenteral administration is generally preferred.
  • Parenteral administration includes nasal administration, subcutaneous injection, intramuscular injection, injection such as intravenous injection, suppository and the like.
  • parenteral administration it can be prepared as a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, and cellulose.
  • the vaccine of the present invention is administered in a therapeutically effective amount.
  • the dose to be administered depends on the subject to be treated and the immune system, and the required dose is determined by the judgment of the clinician. In general, the appropriate dose is 1 to 100 mg of CTL epitope peptide and 10 6 to 10 9 CTL epitope peptide pulsed cells per patient.
  • the administration interval can be set according to the subject and purpose.
  • the CTL epitope peptide of the present invention can be used for the preparation of a passive immunotherapeutic agent.
  • the CTL specific for EBV LMP2 or the CTL specific for EBV EBNA1 obtained as described below can be suspended in human albumin-containing PBS or the like and used as a passive immunotherapy for EBV.
  • the CTL specific for EBV contained in the passive immunotherapeutic agent can be obtained by the following preparation method, and can be purified and used to increase the purity of CTL.
  • PBMC is reacted with an appropriate concentration of EBV-specific MHC-tetramer reagent. Since the EBV-specific CTL bound to the MHC-tetramer reagent is stained with a labeling dye, only the stained CTL is isolated using a cell sorter, a microscope or the like. EBV-specific CTL isolated in this way are T cell stimulating drugs such as anti-CD3 antibodies, PHA, IL-2, and antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Stimulate growth to ensure the number of cells required for passive immunotherapy.
  • T cell stimulating drugs such as anti-CD3 antibodies, PHA, IL-2, and antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Stimulate growth to ensure the number of cells required for passive immunotherapy.
  • CTL preparation method 2 EBV-specific MHC-monomer and / or MHC-tetramer reagent is immobilized on a sterile plate and PBMC is cultured on the immobilized plate.
  • PBMC PBMC is cultured on the immobilized plate.
  • EBV-specific CTL bound to MHC-monomer and / or MHC-tetramer reagent immobilized on the plate other cells floating without being bound are washed off on the plate. Suspend only the specific CTL remaining in the fresh medium.
  • EBV-specific CTLs isolated in this way are stimulated with anti-CD3 antibodies, PHA, IL-2 and other T cell stimulating agents, and antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Proliferate to ensure the number of cells required for passive immunotherapy.
  • CTL preparation method 3 Acts agonistically on EBV-specific MHC-monomers and / or MHC-tetramer reagents and costimulatory molecules such as CD80, CD83, CD86, or CD28, a T cell ligand that binds to costimulatory molecules
  • the antibody to be immobilized is immobilized on a sterile plate, and PBMC is cultured on the immobilized plate.
  • PBMC is cultured on the immobilized plate.
  • IL-2 is added to the medium and cultured in a 5% CO 2 constant temperature bath at 37 ° C. for 7 to 14 days.
  • the cultured cells are collected and cultured on a new solid phase plate. By repeating this operation, CTLs with the number of cells necessary for passive immunotherapy are secured.
  • CTL preparation method 4 Stimulate PBMC or T cells directly with the CTL epitope peptide of the present invention, or with antigen-presenting cells pulsed with the peptide, gene-introduced antigen-presenting cells, or artificially prepared antigen-presenting cells with antigen-presenting ability To do. Stimulation can be in vitro, but may also be in vivo. When stimulated in vitro, CTL induced by stimulation is cultured at 37 ° C for 7 to 14 days in a 5% CO 2 thermostat. In culture, CTL epitope peptide and IL-2, or antigen-presenting cells and IL-2 stimulation are repeated once a week to secure the number of CTLs necessary for passive immunotherapy.
  • a magnetically labeled secondary antibody and a magnetically labeled cell separation device are available from, for example, Dynal and Miltenyi Biotec GmbH.
  • the EBV-specific CTL isolated in this way is stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, IL-2, and the number of cells required for passive immunotherapy is ensured.
  • EBV-specific CTLs can be purified by utilizing cytokines released by EBV-specific CTLs.
  • the cytokine released from the CTL is captured on the cell surface with a specific antibody, stained with an anti-cytokine-labeled antibody, and then magnetically labeled for the labeling substance-specific After reacting with the antibody, it can be purified using a magnetically labeled cell separator.
  • the EBV-specific CTL isolated in this way is stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, IL-2, and the number of cells required for passive immunotherapy is ensured.
  • Cell surface proteins eg CD137, CD107a, CD107b, CD63, CD69, etc.
  • Cell surface proteins eg CD137, CD107a, CD107b, CD63, CD69, etc.
  • CTLs Betts MR , Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA.Sensitive and viable identification of antigen-specific CD8 + T cells by a flow cytometric assay for degranulation.
  • J Immunol Methods.2003; 281: 65-78 Trimble LA, Shankar P, Patterson M, Daily JP, Lieberman J.
  • Human immunodeficiency virus-specific circulating CD8 T lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules for T-cell activation. J Virol. 2000; 74: 7320 -7330).
  • CTL can be similarly purified by magnetically labeling such an anti-IgG antibody against the specific antibody.
  • specific CTLs can be purified by coating these specific antibodies onto a plastic plate for culturing, culturing stimulated PBMC using this plate, and washing away the cell population that did not bind to the plate. It is.
  • the EBV-specific CTL isolated in this way is stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, IL-2, and the number of cells required for passive immunotherapy is ensured.
  • Quantification of EBV-specific CTL Knowing whether EBV-specific CTLs are present in the peripheral blood of cancer patients or the variation in their amounts is important information for predicting EBV-specific CTL epitope peptides. Quantification of EBV-specific CTL can be performed by the following three methods using the CTL epitope peptide of the present invention.
  • Quantitation method 1 CTL specific for EBV in peripheral blood can be quantified using the MHC-tetramer reagent produced using the CTL epitope peptide of the present invention.
  • the quantification can be performed, for example, as follows. Peripheral blood or PBMC are reacted with an appropriate concentration of MHC-tetramer reagent. Since the CTL bound to the MHC-tetramer reagent is stained with a labeling dye, it is counted using a flow cytometer, a microscope or the like. When reacting with MHC-tetramer reagent, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, etc. labeled with a different dye from MHC-tetramer reagent can be reacted to simultaneously determine T cell subsets of EBV-specific CTLs. it can.
  • Quantitation method 2 This is a method for quantifying cytokines and / or chemokines such as IFN ⁇ (interferon gamma), TNF ⁇ (tumor necrosis factor alpha), and interleukin produced by CTL by stimulating PBMC with the CTL epitope peptide of the present invention.
  • IFN ⁇ interferon gamma
  • TNF ⁇ tumor necrosis factor alpha
  • interleukin produced by CTL by stimulating PBMC with the CTL epitope peptide of the present invention.
  • PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 ⁇ 10 6 / mL, and the CTL epitope peptide of the present invention is added. Further, an intracellular protein transport inhibitor (eg, Brefeldin A, Monensin, etc.) is added, and the cells are cultured at 37 ° C. for 5 to 16 hours in a 5% CO 2 constant temperature bath.
  • an intracellular protein transport inhibitor eg, Brefeldin A, Monensin, etc.
  • the cells After culturing, the cells are reacted with a T cell marker antibody (anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody) or MHC-tetramer reagent, and after fixing the cells, membrane permeation treatment is performed, and a dye-labeled anti-IFN ⁇ antibody is reacted. Analysis is performed using a flow cytometer or the like, and the IFN ⁇ positive cell rate in total cells, T cells, or MHC-tetramer reagent positive cells is quantified.
  • a T cell marker antibody anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody
  • MHC-tetramer reagent MHC-tetramer reagent
  • PBMC PBMC are plated on a 96-well MultiScreen-HA plate (Millipore) on which an anti-IFN ⁇ antibody is immobilized. Thereafter, the CTL epitope peptide is placed in each well and cultured in a 5% CO 2 thermostat incubator at 37 ° C. for 20 hours. On the next day, the plate is washed and reacted with anti-IFN ⁇ antibody and peroxidase-labeled anti-IgG antibody in this order. Further, a substrate of peroxidase is added, the IFN ⁇ spot is visualized by color development, and quantified by counting using a stereomicroscope or ELISPOT analyzer (CTL).
  • CTL ELISPOT analyzer
  • PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 ⁇ 10 6 / mL, and the CTL epitope peptide of the present invention is added. Incubate for 24-48 hours at 37 ° C in a 5% CO 2 oven. After the culture, the supernatant is collected, and the IFN ⁇ concentration contained therein is quantified using a commercially available ELISA kit (for example, Quantikine ELISA Human IFN ⁇ Immunoassay from R & D Systems).
  • a commercially available ELISA kit for example, Quantikine ELISA Human IFN ⁇ Immunoassay from R & D Systems.
  • Quantitation method 3 Quantification is performed using cell surface protein specific antibodies.
  • CTLs specific for CTL epitope peptides have been reported to enhance the expression of cell surface proteins (eg, CD137, CD107a, CD107b, CD63, CD69, etc.) by specific stimulation. Therefore, by mixing PBMC stimulated with a CTL epitope peptide and a labeled antibody that specifically recognizes a cell surface protein, CTL binds to the labeled antibody and is stained with a labeled dye. Stained CTL can be counted and quantified using a flow cytometer, a microscope or the like. Furthermore, by adding an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody or the like labeled with a dye different from the labeled antibody, the T cell subset of specific CTL can be determined simultaneously.
  • the number of cells is counted, 10/11 amount of cells are suspended in RPMI1640 medium, and culture plasma is added to a final concentration of 10%.
  • CTL epitope peptide followed, injected 37 ° C. in the culture bag using a luer lock syringe (CultiLife215 TAKARA BIO Inc.), to stand at CO 2 thermostat at 5% CO 2 (CTL induction Bag) .
  • 1/11 amount of cells were suspended in RPMI1640 medium, culture plasma was added to a final concentration of 10%, anti-CD3 antibody was added to a final concentration of 1 ⁇ g / mL, and then a luer lock syringe was used.
  • both antigen-specific CTL and antigen-presenting cells can be induced in about 14 days from the start of culture. Approximately 14 days later, cells in the antigen-presenting cell induction bag are collected, the number of cells is counted, centrifuged, suspended in RPMI1640 medium, CTL epitope peptide is added, and cultured at room temperature for 1 hour. After centrifugation, the supernatant is removed by suction and suspended in a medium containing IL-2 (AlyS505N IL-2 100 IU / mL 10% plasma) (peptide pulse antigen-presenting cells). Similarly, the cells in the CTL induction bag are collected, counted and centrifuged, and the supernatant is removed by aspiration.
  • IL-2 AlyS505N IL-2 100 IU / mL 10% plasma
  • the cells are suspended in an IL-2 containing medium (AlyS505N IL-2 100 IU / mL 10% plasma).
  • Peptide pulse antigen-presenting cells in an amount equal to the number of cells in the CTL induction bag are mixed and injected into a culture bag using a luer lock syringe. Thereafter, the culture bag is transferred to a CO 2 thermostatic bath at 37 ° C. and 5% CO 2 to start the culture.
  • the growth medium (AlyS505N IL-2 1000 IU / mL) is added to the culture bag. After culturing for 3 days in a CO 2 thermostatic bath, the growth medium is added to the culture bag. Thereafter, a growth medium is added every two days.
  • the CTL epitope candidate peptide specific for CKAP4 of the present invention is a peptide comprising 8 to 12 amino acids having a binding motif for the target HLA class I molecule with respect to the amino acid sequence of the CKAP4 protein.
  • Multiple software published on the Internet that can be searched (Lundegaard C, Lund O, Buus S, Nielsen M, Major histocompatibility complex class I binding predictions as a tool in epitope discovery. Immunology., 2010; 130: 309- 318) can be selected.
  • HLA class I molecules mainly include HLA-A, HLA-B, and HLA-C, and the epitope peptides that are displayed by binding to these consist of 8 to 10 amino acids.
  • the second, ninth or tenth amino acid from the N-terminal side of the epitope peptide is the most important amino acid for binding to the HLA class I molecule, and is called an anchor motif. It has been reported that this anchor motif varies depending on the type of each HLA class I molecule. For example, as a peptide that binds to the HLA-A2 molecule, the most studied in the world, Leu is placed at the second position from the N-terminus, and Leu or Val is placed at the 9th or 10th position.
  • the CTL epitope candidate peptides shown in SEQ ID NOs: 36 to 47 of the present invention can be prepared by various conventional peptide synthesis methods.
  • organic chemical synthesis methods such as solid phase peptide synthesis methods, or nucleic acids encoding peptides can be prepared and prepared using recombinant DNA technology.
  • combination by a commercially available chemical synthesizer is also possible.
  • the CTL epitope is composed of peptide fragments having a length of 8 to 12 amino acid residues, and these are produced by various degradation of antigen proteins in cells. Specifically, the antigen protein is first degraded by the proteasome in the cytoplasm to form the C-terminus of the peptide. Thereafter, the peptide fragment is transported to the endoplasmic reticulum lumen by a TAP (transporter-associated-with-antigen-processing) molecule, where it becomes a peptide fragment of 8 to 12 amino acid residues only after the N-terminus is formed by the protease ERAP1.
  • TAP transporter-associated-with-antigen-processing
  • candidate peptides obtained using the prediction software include those that cannot actually constitute peptide fragments having a length of 8 to 12 amino acid residues in the cell.
  • T cells that react strongly with self-antigen-derived peptide fragments induce apoptosis in the thymus by negative selection and are eliminated.
  • T cells that react with foreign antigens are selected by positive selection. From such a T cell differentiation process, T cells that react with a self-antigen-derived peptide fragment are usually removed by the thymus, and it is thought that only a small part is present in peripheral blood.
  • peptide fragments derived from autoantigens are presented on HLA molecules expressed on the membrane surface of all nucleated cells and platelets, and there are innumerable peptide fragments that bind to HLA.
  • CTL recognizes only a part of the peptide fragments derived from HLA on the cell membrane surface that are recognized by HLA, and most of the CTL epitope candidate peptides by the prediction software are not recognized by CTL. It is believed that. That is, it is considered that there is no antigenicity.
  • obtaining a CTL epitope candidate peptide by simply using prediction software is greatly different from identifying a CTL epitope peptide responsible for an immune response.
  • the inventors have identified a CTL epitope peptide by a method of directly detecting in vivo CTL specific for a candidate peptide using an MHC-tetramer reagent.
  • the detection of specific CTLs from a sample such as peripheral blood by the MHC-tetramer reagent means that a candidate peptide-specific immune response has been induced in vivo, and the candidate peptide is a CTL epitope. Indicates that it is a peptide.
  • the CTL epitope candidate peptide selected by the above-described method can be a CKAP4-specific CTL epitope peptide only after the following studies (1) to (3).
  • a TAP gene-deficient cell line deficient in this TAP molecule cannot express peptide fragments, which are degradation products of endogenous proteins, on the cell membrane surface.
  • human lymphoblastoid cell line T2 which is a typical TAP gene-deficient cell line, or a cell line in which HLA-A24 molecule is introduced into T2 (T2-A24) is expressed on the cell membrane surface. Very unstable. However, when bound to an externally supplied peptide, the HLA molecule is stabilized on the cell membrane surface. Using this property, the TAP gene-deficient cell line can be used in experiments for verifying the binding property between HLA molecules and externally supplied peptides.
  • the target HLA molecule and The binding of CTL epitope candidate peptides can be examined.
  • the CTL epitope candidate peptide added to the HLA molecule expressed by the TAP gene-deficient cell line binds, the HLA molecule-peptide complex is stabilized on the cell membrane surface, and when stained with an anti-HLA antibody, the expression of the HLA molecule is enhanced. Is observed.
  • MHC-tetramer reagent is a ternary complex (MHC-monomer) consisting of MHC (HLA for humans), ⁇ 2-microglobulin and peptide fragments in vitro. It is an incorporated reagent.
  • the MHC-tetramer reagent is the only reagent that can selectively detect antigen-specific CTLs that are MHC restricted.
  • MHC-tetramer reagent can not only quantify the amount of CTL by analyzing with flow cytometry after co-staining with anti-CD (cluster of differentiation) antibody, anti-cytokine antibody, etc., but also its activation state and differentiation stage It is possible to evaluate each cell individually.
  • the first step in MHC-tetramer reagent production begins with folding where the raw materials MHC, ⁇ 2-microglobulin and peptide are mixed in a suitable solution in a test tube. In the folding solution, a three-component complex (MHC-monomer) is formed by the association reaction of these three raw materials.
  • the used CTL epitope candidate peptide can be identified as an antigenic peptide (epitope peptide) having CTL inducing ability.
  • T cells naive T cells
  • T cells transferred from the thymus are activated and differentiated into effector T cells only upon antigen stimulation by dendritic cells, macrophages and other antigen-presenting cells.
  • naive T cells in peripheral blood were differentiated because they did not use artificially prepared antigen-presenting cells. It is thought that effector / memory T cells are proliferating by peptide stimulation.
  • MHC-monomer and MHC-tetramer reagent using a CKAP4-specific CTL epitope candidate peptide can be prepared by a known method (US Patent Number 5,635,363, French Application Number FR9911133).
  • An MHC-monomer that is a complex of three of a HLA class I molecule purified from a recombinant host for protein expression, ⁇ 2-microglobulin, and a CKAP4-specific CTL epitope candidate peptide of the present invention is formed in a folding solution.
  • a biotin binding site is added to the C-terminus of the recombinant HLA class I molecule in advance, and biotin is added to this site after MHC-monomer formation.
  • a MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4. It can also be used in combination with antibodies against cell surface proteins (CD62L, CCR7, CD45RA, etc.) to examine the differentiation stage of CTL (Seder RA, Ahmed R. Similarities and differences in CD4 + and CD8 + effector and memory T cell generation) Nat Immunol. 2003; 4: 835-842).
  • CTL against HCV Hepatitis C virus
  • HCV Hepatitis C virus
  • CTL against HCV exists as one of the causes for maintaining persistent infection, but CTL does not produce cytokines, or the proportion of CTL produced is extremely low.
  • anomaly Gaener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, Walker B, Sullivan J, Phillips R, Pape GR , Klenerman P. Sustained dysfunction of antiviral CD8 + T lymphocytes after infection with hepatitis C virus. J Virol. 2001; 75: 5550-5558).
  • HCMV-specific CTL after bone marrow transplantation it is considered effective not only to examine the presence or absence of CTL but also to examine the strength of cytokine production in order to measure the timing of administration of antiviral drugs, etc.
  • Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8 + T cells.Blood. 2002; 100: 3690-3697).
  • the CTL epitope peptide of the present invention can be used as a peptide vaccine in active immunotherapy. That is, a vaccine comprising the CTL epitope peptide of the present invention is administered to a patient, and CKAP4-specific CTL is proliferated in the body, so that treatment for malignant tumor can be expected.
  • Vaccine using antigen-presenting cell The antigen-presenting cell on which the CTL epitope peptide of the present invention is presented can be used as a vaccine in active immunotherapy.
  • Antigen-presenting cells with CTL epitope peptides presented 1.
  • CTL epitope peptide pulse antigen-presenting cells in which antigen-presenting cells and CTL epitope peptides are mixed for 30 minutes to 1 hour in an appropriate culture medium.
  • the antigen-presenting cell means, for example, a dendritic cell, a B cell, a macrophage, a certain type of T cell, etc., and is a cell that expresses an HLA molecule to which the peptide can bind on its cell membrane surface, It means something that has stimulating ability.
  • Artificial antigen-presenting cells with antigen-presenting ability are artificially prepared by immobilizing a ternary complex of HLA molecule, CTL epitope peptide and ⁇ 2-microglobulin on beads such as lipid bilayer membrane, plastic or latex.
  • costimulatory molecules such as CD80, CD83, and CD86 that can stimulate CTLs
  • fix antibodies that act agonistically on CD28, a T cell ligand that binds to costimulatory molecules.
  • Cutting edge predetermined avidity of human CD8 T cells expanded on calibrated MHC / anti -CD28-coated microspheres. J Immunol. 2003; 171: 4974-49 78, Oosten LE, Blokland E, van Halteren AG, Curtsinger J, Mescher MF, Falkenburg JH, Mutis T, Goulmy E. Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes.Blood. 2004; 104: 224 -226).
  • the nucleic acid of the CTL epitope peptide of the present invention can be used for DNA vaccines, recombinant virus vector vaccines and the like in active immunotherapy.
  • it is desirable to change the nucleic acid sequence of the CTL epitope peptide to codon usage suitable for the host producing the recombinant vaccine or the recombinant virus vaccine (Casimiro, DR et al. Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid , Recombinant Vaccinia Virus, and Replication-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene. J.
  • a vaccine comprising a CTL epitope peptide of the present invention or an antigen-presenting cell on which a CTL epitope peptide is presented can be prepared using methods known in the art.
  • a vaccine includes an injection or a solid preparation containing the CTL epitope peptide of the present invention as an active ingredient.
  • the CTL epitope peptide can be formulated in a neutral or salt form.
  • pharmaceutically acceptable salts include inorganic salts such as hydrochloric acid and phosphoric acid, and organic acids such as acetic acid and tartaric acid. .
  • the antigen-presenting cell on which the CTL epitope peptide of the present invention is presented is a pharmaceutically acceptable excipient compatible with the peptide or the activity of the cell, such as water, saline, dextrose, ethanol, It can be used by mixing with glycerol, DMSO (dimethyl sulphoxide), other adjuvants, or a combination thereof. Furthermore, you may add adjuvants, such as albumin, a wetting agent, and an emulsifier, as needed.
  • the vaccine of the present invention can be administered by parenteral administration or oral administration, but parenteral administration is generally preferred.
  • parenteral administration include nasal administration, subcutaneous / intradermal injection, intramuscular injection, intravenous injection and other suppositories, and suppositories.
  • oral administration it can be prepared as a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, and cellulose.
  • the vaccine of the present invention is administered in a therapeutically effective amount.
  • the dose to be administered depends on the subject to be treated and the immune system, and the required dose is determined by the judgment of the clinician. In general, the appropriate dose is 1 to 100 mg of CTL epitope peptide and 10 6 to 10 9 CTL epitope peptide pulsed cells per patient.
  • the administration interval can be set according to the subject and purpose.
  • the CTL epitope peptide of the present invention can be used for the preparation of a passive immunotherapeutic agent.
  • the CTL specific for CKAP4 obtained as described below can be suspended in human albumin-containing PBS or the like and used as a passive immunotherapeutic agent for malignant tumors expressing CKAP4.
  • CTL specific for CKAP4 contained in the passive immunotherapeutic agent can be obtained by the following preparation method, and can be purified and used to increase the purity of CTL.
  • CTL preparation method 1 PBMC is reacted with an appropriate concentration of CKAP4-specific MHC-tetramer reagent. Since the CKAP4-specific CTL bound to the MHC-tetramer reagent is stained with the labeling dye, only the stained CTL is isolated using a cell sorter, a microscope or the like. CTLs specific for CKAP4 isolated in this way are anti-CD3 antibodies, PHA, IL-2 and other T cell stimulating agents, and antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Stimulate growth to ensure the number of cells required for passive immunotherapy.
  • CTL preparation method 2 CKAP4-specific MHC-monomer and / or MHC-tetramer reagent is immobilized on a sterile plate and PBMC is cultured on the immobilized plate.
  • PBMC is cultured on the immobilized plate.
  • CKAP4-specific CTL bound to the MHC-monomer and / or MHC-tetramer reagent immobilized on the plate after washing off other cells that are not bound, Only the remaining CKAP4-specific CTL is suspended in fresh medium.
  • CKAP4-specific CTL isolated in this way is stimulated to proliferate on T-cell stimulating drugs such as anti-CD3 antibodies, PHA, IL-2, and antigen-presenting cells whose proliferation ability has been lost by treatment with X irradiation or mitomycin. And secure the number of cells required for passive immunotherapy.
  • T-cell stimulating drugs such as anti-CD3 antibodies, PHA, IL-2, and antigen-presenting cells whose proliferation ability has been lost by treatment with X irradiation or mitomycin. And secure the number of cells required for passive immunotherapy.
  • CTL preparation method 3 Acts agonistically on CKAP4-specific MHC-monomers and / or MHC-tetramer reagents and costimulatory molecules such as CD80, CD83, CD86, or CD28, a T cell ligand that binds to costimulatory molecules
  • the antibody to be immobilized is immobilized on a sterile plate, and PBMC is cultured on the immobilized plate.
  • IL-2 is added to the medium and cultured in a 5% CO 2 constant temperature bath at 37 ° C for 7 to 10 days.
  • the cultured cells are collected and cultured on a new solid phase plate. By repeating this operation, CTLs with the number of cells necessary for passive immunotherapy are secured.
  • CTL preparation method 4 Stimulate PBMC or T cells directly with the CTL epitope peptide of the present invention, or with antigen-presenting cells pulsed with the peptide, gene-introduced antigen-presenting cells, or artificially prepared antigen-presenting cells with antigen-presenting ability To do. Stimulation can be in vitro, but may also be in vivo. When stimulated in vitro, CTL induced by stimulation is cultured at 37 ° C for 7 to 14 days in a 5% CO 2 thermostat. In culture, CTL of the number of cells necessary for passive immunotherapy is ensured by repeating stimulation with CTL epitope peptide and IL-2 or antigen-presenting cells and IL-2 once a week.
  • CKAP4-specific MHC-tetramer reagent reacts with CTL induced by the CTL preparation method, and a secondary antibody magnetically labeled with an antibody against a labeled dye labeled with MHC-tetramer reagent And can be separated.
  • a magnetically labeled secondary antibody and a magnetically labeled cell separation device are available from, for example, Dynal and Miltenyi Biotec GmbH.
  • the CKAP4-specific CTL isolated in this manner is stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, IL-2, etc., and the number of cells necessary for passive immunotherapy is ensured.
  • Purification by secreted cytokine CKAP4-specific CTLs can be purified by using cytokines released by CKAP4-specific CTLs.
  • cytokines released by CKAP4-specific CTLs For example, by using a kit available from Miltenyi Biotec GmbH, the cytokine released from CTL is supplemented with a specific antibody on the cell surface, stained with a cytokine-specific labeled antibody, and then magnetically labeled labeling substance specific It is also possible to purify using a magnetically labeled cell separation apparatus after reacting with a typical antibody.
  • the CKAP4-specific CTL isolated in this manner is stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, IL-2, etc., and the number of cells necessary for passive immunotherapy is ensured.
  • Cell surface proteins eg CD137, CD107a, CD107b, CD63, CD69, etc.
  • Cell surface proteins eg CD137, CD107a, CD107b, CD63, CD69, etc.
  • CTLs Betts MR , Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA.Sensitive and viable identification of antigen-specific CD8 + T cells by a flow cytometric assay for degranulation.
  • J Immunol Methods.2003; 281: 65-78 Trimble LA, Shankar P, Patterson M, Daily JP, Lieberman J.
  • CKAP4-specific CTL can be similarly purified by magnetically labeling such an anti-IgG antibody against the specific antibody.
  • these specific antibodies can be coated on a plastic plate for culture, and PBMCs can be cultured using this plate, and the CKAP4-specific CTL can be purified by washing away the cell population that did not bind to the plate. Is possible.
  • the CKAP4-specific CTL isolated in this manner is stimulated and proliferated with a T cell stimulating agent such as anti-CD3 antibody, PHA, IL-2, etc., and the number of cells necessary for passive immunotherapy is ensured.
  • Quantitation method 1 CTL specific for CKAP4 in peripheral blood can be quantified using the MHC-tetramer reagent produced using the CTL epitope peptide of the present invention.
  • the quantification can be performed, for example, as follows. Peripheral blood or PBMC are reacted with an appropriate concentration of MHC-tetramer reagent. Since the CTL bound to the MHC-tetramer reagent is stained with a labeling dye, it is counted using a flow cytometer, a microscope or the like. When reacting with MHC-tetramer reagent, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, etc. labeled with a different dye from MHC-tetramer reagent can be reacted to simultaneously determine T cell subsets of CKAP4-specific CTL it can.
  • Quantitation method 2 This is a method for quantifying cytokines and / or chemokines such as IFN ⁇ (interferon gamma), TNF ⁇ (tumor necrosis factor alpha), and interleukin produced by CTL by stimulating PBMC with the CTL epitope peptide of the present invention.
  • IFN ⁇ interferon gamma
  • TNF ⁇ tumor necrosis factor alpha
  • interleukin produced by CTL by stimulating PBMC with the CTL epitope peptide of the present invention.
  • PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 ⁇ 10 6 / mL, and the CTL epitope peptide of the present invention is added. Furthermore, an intracellular protein transport inhibitor (for example, Brefeldin A, Monensin, etc.) is added, and cultured at 37 ° C. for 5 to 16 hours in a 5% CO 2 thermostat.
  • an intracellular protein transport inhibitor for example, Brefeldin A, Monensin, etc.
  • the cells After culturing, the cells are reacted with a T cell marker antibody (anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody) or MHC-tetramer reagent, and after fixing the cells, membrane permeation treatment is performed, and a dye-labeled anti-IFN ⁇ antibody is reacted. Analysis is performed using a flow cytometer or the like, and the IFN ⁇ positive cell rate in total cells, T cells, or MHC-tetramer reagent positive cells is quantified.
  • a T cell marker antibody anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody
  • MHC-tetramer reagent MHC-tetramer reagent
  • PBMC PBMC are plated on a 96-well MultiScreen-HA plate (Millipore) on which an anti-IFN ⁇ antibody is immobilized. Thereafter, the CTL epitope peptide is placed in each well and cultured in a 5% CO 2 thermostat incubator at 37 ° C. for 20 hours. On the next day, the plate is washed and reacted with anti-IFN ⁇ antibody and peroxidase-labeled anti-IgG antibody in this order. Further, a substrate of peroxidase is added, the IFN ⁇ spot is visualized by color development, and quantified by counting using a stereomicroscope or ELISPOT analyzer (CTL).
  • CTL ELISPOT analyzer
  • PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 ⁇ 10 6 / mL, and the CTL epitope peptide of the present invention is added. Incubate for 24-48 hours at 37 ° C in a 5% CO 2 oven. After the culture, the supernatant is collected, and the IFN ⁇ concentration contained therein is quantified using a commercially available ELISA kit (for example, Quantikine ELISA Human IFN ⁇ Immunoassay from R & D Systems).
  • a commercially available ELISA kit for example, Quantikine ELISA Human IFN ⁇ Immunoassay from R & D Systems.
  • Quantitation method 3 Quantification is performed using cell surface protein specific antibodies.
  • CTL specific for CTL epitope peptide has been reported to increase the expression of cell surface proteins (eg CD137, CD107a, CD107b, CD63, CD69, etc.) by specific stimulation (Watanabe K, Suzuki S, Kamei M , Toji S, Kawase T, Takahashi T, Kuzushima K, and Akatsuka Y. CD137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adoptive immunotherapy. Int J Hematol, 2008; 88; 311-320).
  • CTL binds to the labeled antibody and is stained with a labeled dye. Stained CTL can be counted and quantified using a flow cytometer, a microscope or the like. Furthermore, by adding an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody or the like labeled with a dye different from the labeled antibody, the T cell subset of specific CTL can be determined simultaneously.
  • pMHC antibody The identified monoclonal antibody specific for the complex of cancer antigen epitope peptide and MHC (hereinafter referred to as pMHC antibody) can specifically detect cancer cells presenting the epitope peptide on the surface of the cell membrane. For this reason, pMHC antibodies can be used as diagnostic agents for cancer immunotherapy, and can also be used as highly specific therapeutic antibodies by binding antibody-dependent cytotoxicity (ADCC activity) or anticancer drugs. There is utility. In general, pMHC antibodies are obtained by the phage display method.
  • the phage display method is a system for expressing a foreign gene as a fusion protein so as not to lose the infectivity of the phage.
  • a cancer antigen epitope peptide and MHC complex (MHC-monomer) is immobilized on an ELISA plate or immobilized with a biotin-avidin bond, and this is reacted with a phage library.
  • MHC-monomer a cancer antigen epitope peptide and MHC complex
  • the obtained antibody can be evaluated by adding an epitope peptide to the aforementioned TAP gene-deficient strain T2, reacting the antibody, and measuring the average fluorescence intensity with FCM.
  • EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise noted, the experimental methods were conducted with reference to the book (Immune Experiment Method, Shunsuke Ueda, Susumu Hamada, Honjo, Toshiyuki Sasaoka, Nanedo 1995).
  • Example 1 [Selection of EBV-specific CTL epitope candidate peptides] EBV is classified into type 1 and type 2 depending on the difference in the expressed protein. Type 1 infections, including AG876, are rare in Asia, most of which are infected / hidden in humans (Dambaugh T, Hennessy K, Chamnankit L, Kieff E.
  • Epstein-Barr DNA may encode Epstein-Barr nuclear antigen 2. Proc Natl Acad Sci US A. 1984 Dec; 81 (23): 7632-6). However, cases of co-infection of EBV in the same individual have been reported, and race- and region-dependent diversity of EBV-infected strains have been reported (Apolloni A, Sculley TB. Detection of A-type and B- type Epstein-Barr virus in throat washings and lymphocytes.Virology.
  • Selection of LMP2 and EBNA1-specific CTL epitope candidate peptides of the present invention is based on the amino acid sequence of B95.8 (GenBank: V01555.2), the most representative strain belonging to EBV type 1, and other strains. This was carried out with reference to amino acid mutations in. Specifically, it was carried out by collating with a plurality of software published on the Internet, which can search for a CTL epitope candidate peptide consisting of 8 to 10 amino acids having a binding motif for the HLA-A11 molecule.
  • CTL epitope candidate peptides consisting of 9 or 10 amino acids having a binding motif of HLA-A11 molecule from the amino acid sequences of EBVEBLMP2 and EBNA1 were selected, a total of 33 types including 22 types for LMP2 and 11 types for EBNA1. These peptides were synthesized and shown below as CTL epitope candidate peptides. Amino acid mutation sites in different EBV strains are underlined. In the present invention, the B95.8 strain is a wild strain, and amino acid substitutions in other strains are called mutations.
  • HLA-A * 11 01 restricted epitope peptide (ATVQGQNLK, SEQ ID NO: 34) derived from pp65 protein of human cytomegalovirus (CMV) was synthesized.
  • CMV human cytomegalovirus
  • survivin-2B HLA-A * 24: 02 restricted epitope peptide AYACNTSTL, SEQ ID NO: 35 was synthesized.
  • Tables 1, 2, and 3 show the characteristics of the synthesized LMP2 and EBNA1 HLA-A11: 01 restricted CTL epitope peptides and the peptides synthesized for control.
  • the peptide name is indicated by three amino acid sequences from the N-terminal side of the synthesized peptide.
  • peptide name (a in the table), amino acid sequence (b in the table), position on the amino acid sequence of the derived protein, number of amino acids, NetMHC3.4 used for analysis (http://www.cbs.dtu.dk / Services / NetMHC /) HLA Peptide Binding Predictions (Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, Brunak S, Lund O. Reliable prediction vel ations-no . Protein Sci. 2003 May; 12 (5): 1007-17, Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M.
  • NetMHC-3.0 accurate web accessible predictions of human, mouse HC I affinities for peptides of length 8-11. Nucleic Acids Res. 2008 Jul 1; 36 (Web Server issue): W509-12., Lundegaard C, Lund O, Nielsen M. Accurate approximation method for predictionforHC peptides of length 8, 10 and 11 using prediction tools tra Ined (on 9mers. Bioinformatics. 2008 Jun 1; 24 (11): 1397-8.), the scores (c in the table) are shown in descending order. This score is a numerical value that predicts the affinity between HLA-A11 and the peptide. The higher the score, the more likely the HLA and peptide may form a stable complex.
  • the scores of NetMHC 3.4 shown in Table 1, Table 2 and Table 3 are shown as representative examples obtained with 11 types of analysis software used in the analysis.
  • [Folding test of EBV-specific CTL epitope candidate peptide] The inventors conducted a folding test using 35 types of peptides described in Tables 1, 2 and 3. Specifically, HLA-A * 11: 01, ⁇ 2-microglobulin expressed using E. coli expression system and ⁇ 2-microglobulin, and synthetic peptide were added to the folding solution, mixed, and the folding solution was collected over time. The analysis was performed with a gel filtration column.
  • MHC-monomer When gel filtration column analysis shows the formation of a ternary complex (MHC-monomer) of HLA-A * 11: 01, ⁇ 2-microglobulin, and EBV-specific CTL epitope candidate peptide, MHC-monomer Since the molecular weight is large, the elution time in the gel filtration column analysis is shortened. Further, the amount of MHC-monomer formation can be calculated from the peak area obtained by the absorption wavelength of 280 nm. On the other hand, MHC-monomer formation is not confirmed with candidate peptides that do not bind to HLA molecules. A typical gel filtration column analysis example in which MHC-monomer formation is observed is shown in FIG.
  • HLA molecules and ⁇ 2-microglobulin were solubilized in 8M urea after purification of the insoluble fraction as inclusion bodies when purified using the E. coli expression system. Soluble HLA molecules that do not lead to MHC-monomer formation are detected as aggregates in 7-8 minutes. However, most of the aggregates are removed by filter filtration, which is a pretreatment step of gel filtration column analysis.
  • ⁇ 2-microglobulin is a soluble protein, solubilized in a folding solution, and is detected in the vicinity of 14 minutes when using Superdex 75GL10 / 300GL (GE Healthcare). After 15 minutes, the folding solution composition and peptide are detected. The peak of MHC-monomer is not confirmed immediately after the start of the folding test (day 0), but the peak increases after 1 day (day 1) and 3 days (day (3), and MHC-monomer formation is progressing smoothly. Shows things.
  • HLA-A * 11 01 restricted epitope peptide derived from CMV pp65 protein (ATVQGQNLK, SEQ ID NO: 34)
  • survivin-2B derived HLA-A * 24 02 restricted epitope peptide ( AYACNTSTL, SEQ ID NO: 35) was used for comparison.
  • the area of the peak indicating MHC-monomer formation is shown as a bar graph.
  • Tables 4 and 5 show the results of comparing SEQ ID NOs: 1 to 22 (LMP2) and SEQ ID NOs: 23 to 33 (EBNA1) with SEQ ID NOs: 34 to 35, which are positive control peptides and negative controls, respectively.
  • Tables 4 and 5 show HLA-A * 11: 01 binding properties of peptide candidates.
  • the presence / absence of binding is indicated by ⁇ when the value exceeding the highest binding value of the negative control (135,674 ⁇ V * sec) is considered as having a binding property, and by ⁇ when the lower value is not binding.
  • the HLA-A * 11: 01 binding determined by the experimental values does not necessarily match the predicted value obtained by the computer algorithm, and the experimental values of the folding test are the candidate peptides and HLA-A in in vitro.
  • * Represents the binding with 11:01.
  • the results of the folding test showed HLA-A * 11: 01 binding for all of the lower eight peptide candidates predicted by the computer algorithm (predicted value ⁇ 0.400).
  • the predicted values of the four peptides that do not show the HLA-A * 11: 01 binding property as a result of the folding test are as follows. VML: 0.417; LVL (9mer): 0.439; TTM: 0.477; KIL: 0.602. It can be seen that although neither predicted value is higher than 0.400, it does not bind to HLA-A * 11: 01. Thus, the binding between the epitope candidate peptide and the HLA molecule cannot be judged only by the computer algorithm.
  • EBV LMP2-specific MHC-tetramer reagent Based on the results of the folding test, a PE-labeled MHC-tetramer reagent was prepared using HLA-A * 11: 01-binding EBV LMP2-specific or EBV EBNA1-specific CTL epitope candidate peptides.
  • the MHC-tetramer reagent produced in the present invention is abbreviated as, for example, ASS (10mer) -Tet, which is HLA-A * 11: 01, ASS (10mer) peptide (ASSYAAAQRK, SEQ ID NO: 1) and ⁇ 2 -Indicates one produced using a microglobulin ternary complex.
  • MHC-monomer which is a complex of HLA class I molecule purified from a recombinant host for protein expression, ⁇ 2-microglobulin and EBV LMP2 or EBV EBNA1-specific CTL epitope candidate peptide of the present invention in an appropriate folding solution To form.
  • a biotin binding site is added to the C-terminus of the recombinant HLA class I molecule in advance, and biotin is added to this site after MHC-monomer formation.
  • a MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4.
  • EBV-specific CTL epitope peptide [Identification of EBV-specific CTL epitope peptide] (Selection of specimen HLA type) As candidate peptides for EBV-specific CTL epitopes, 33 types of candidate peptides having a binding motif for HLA-A * 11: 01 were selected. Furthermore, a folding test revealed that HLA-A * 11: 01, ⁇ 2-microglobulin and 29 EBV-specific CTL epitope candidate peptides bind to each other to form MHC-monomers in vitro.
  • EBV-infected B cell line Kuzushima K, Yamamoto M, Kimura H, Ando Y, Kudo T, Tsuge I, Morishima T.
  • EBV Epstein-Barr virus
  • EBV-infected LCL EBV-infected B cell line
  • NIH-CD40L NIH3T3 cells
  • NIH-CD40L NIH3T3 cells
  • NIH-CD40L inhibited growth by 96 Gy X-ray irradiation and repeated co-culture every 3-4 days
  • Kiem HP Kiem HP
  • Obata Y Morishima Y
  • Kuzushima K Tanimoto M
  • Harada M Takahashi T
  • Akatsuka Y Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells. J Immunol. 2002; 169: 2164-2171).
  • HLA molecule expression and CD80, CD83 and CD86 expression were confirmed.
  • PBMC dendritic cells
  • GM-CSF and IL-4 were added thereto, and cultured for 24 hours.
  • TNF ⁇ , IL-1 ⁇ and PGE2 Prostaglandin E2 were added, and cultured for 24 to 48 hours.
  • the cells recovered by washing gently with an appropriate medium were used as dendritic cells (Kondo E, Topp MS, Kiem HP, Obata Y, Morishima Y, Kuzushima K, Tanimoto M, Harada M, Takahashi T, Akatsuka Y.
  • EBV-specific CTL 1) Induction using antigen-presenting cells
  • the above-described antigen-presenting cells (EBV-infected LCL, CD40-B cells, dendritic cells) were prepared in advance from PBMC holding HLA-A * 11: 01.
  • Antigen-presenting cells are suspended in pulse medium (0.1% human serum albumin / 55 ⁇ M 2-mercaptoethanol / RPMI 1640) or AIM-V medium (Invitrogen), and CTL epitope candidate peptides are added at a concentration of 10 ⁇ g / mL.
  • peptide pulse antigen-presenting cell Peptide pulse antigen-presenting cells were treated with a lethal dose of X-ray irradiation or mitomycin C treatment to lose their ability to grow.
  • This induction method is a method for inducing CTL by introducing a peptide into a PBMC culture solution. It is considered that the peptide is presented to antigen-presenting cells present in PBMC, such as dendritic cells, B cells, macrophages, and certain T cells, and CTL precursor cells contained in PBMC are stimulated to proliferate. Unlike the above-described induction method using antigen-presenting cells, the method is distinguished in that it is not necessary to prepare antigen-presenting cells in advance and can be easily carried out. It is a system that stimulates and proliferates memory / effector type CTL circulating in peripheral blood without using antigen presenting cells.
  • Peripheral blood collected from healthy adults holding HLA-A * 11: 01 was centrifuged at 3,000 rpm for 5-10 minutes, and the plasma portion of the supernatant was collected.
  • PBMCs were separated according to the conventional method except for the plasma portion. It is characterized by adding several percent of plasma to the medium used for induction. In the present invention, good results were obtained when 5% plasma was added.
  • the medium is generally supplemented with appropriate additives and antibiotics for the medium used for cell culture.
  • RPMI1640 Hepes modify Sigma was added with 2-mercaptoethanol, L-glutamine, and antibiotics streptomycin and penicillin.
  • PBMCs were suspended in 1 to 2.5 mL of medium.
  • the concentration of the peptide can be varied depending on the solubility of the peptide. In the present invention, it was carried out at 10 ⁇ g / mL.
  • IL-2 was added at a final concentration of 20-100 U / mL.
  • a polypropylene 14 mL round-bottom tube (BD Biosciences) or 96-well U-bottom cell culture is used.
  • a micro test plate (BD Biosciences) was used. Confirmation of EBV-specific CTL was performed after 2 weeks and 4 weeks of culture. After 2 weeks of culture, stimulation with 10 ⁇ g / mL peptide was performed again. When induction of EBV-specific CTL was confirmed, stimulation was performed using peptide pulse antigen-presenting cells to establish a CTL line.
  • FIG. 4 shows the results of examining specific CTLs using a positive control peptide by an induction method that does not use antigen-presenting cells and examined by an intracellular IFN ⁇ -producing cell quantification method.
  • PBMC isolated from healthy adult peripheral blood that retains HLA-A * 11: 01 but does not retain HLA-A * 24: 02 is an epitope peptide derived from HLA-A * 11: 01-restricted CMV pp65 (ATVQGQNLK, SEQ ID NO: 34)
  • stimulation with HLA-A * 24: 02-restricted survivin-2B-derived epitope peptide AYACNTSTL, SEQ ID NO: 35
  • the result of triple staining with PE-labeled MHC-tetramer reagent (MBL), PC5-labeled anti-CD8 antibody and FITC-labeled anti-IFN ⁇ antibody, and analysis using a flow cytometer are shown.
  • the numbers in the dot plot development view indicate the ratio (%) of the cells present in the four divided regions to the whole living cells.
  • the four-divided area will be referred to as UL (upper left), UR (upper right), LL (lower left), and LR (lower right).
  • IFN ⁇ -producing cells were induced by restimulation in PBMC cultured with the HLA-restricted CTL epitope peptide retained by the donor, and these cells were stained with MHC-tetramer reagent. It is clear that the CTL is specific to the added CTL epitope peptide. Whether or not specific CTLs are induced in this way can also be determined by an intracellular IFN ⁇ -producing cell quantification method. Similarly, an investigation was performed on five healthy adults to determine whether IFN ⁇ -producing cells were induced using EBV LMP2-specific CTL epitope candidate peptides. The ratio of live IFN ⁇ -producing cells present in CD8-positive cells is quantified and shown in FIG. FIG. 6 shows representative results of quantifying intracellular IFN ⁇ -producing cells.
  • peripheral blood of five healthy adults (donor ID numbers: * 11-13, * 11-16, * 11-8, * 11-11, * 11-2) was used, and 21 types of peptides were used. Intracellular IFN ⁇ producing cells were quantified. The percentage (%) of the number of CD8 positive IFN ⁇ positive cells in PBMC is shown on the X axis.
  • Peptide candidates that showed a positive result compared with the negative control are ASS (10mer) (SEQ ID NO: 1) and the positive control peptide CMV pp65. The detailed result of the peptide which showed positive is demonstrated in FIG.
  • Fig. 6 is a dot plot development showing the fluorescence intensity for CD8 on the X-axis and IFN ⁇ on the Y-axis on a log scale, and quantifies intracellular IFN ⁇ -producing cells by restimulation with the same peptide used for induction. The results were shown. The ratio (%) of the number of CD8-positive IFN ⁇ -positive cells to PBMC in UR is shown as a numerical value.
  • peripheral blood To 200 ⁇ L of collected peripheral blood, 10 ⁇ L of PE-labeled MHC-tetramer reagent, 20 ⁇ L of FITC-labeled anti-T cell surface antibody (eg, CD8, CD4, CD3) and the like were added. Furthermore, an anti-CD45 antibody labeled with PC5 or the like may be added in order to exclude non-specific fluorescence due to mixed red blood cells. Gently mixed and left at room temperature for 30 minutes. OptiLyse B (Beckam coulter) was added, and hemolysis fixation was performed according to the instructions. After adding 2 mL of PBS and stirring, it was centrifuged at 400 ⁇ g for 5 minutes. After discarding the supernatant by aspiration, the cells were resuspended in 500 ⁇ L of PBS and analyzed with a flow cytometer within 24 hours.
  • FITC-labeled anti-T cell surface antibody eg, CD8, CD4, CD3
  • CTL lines derived using PBMC or CTL epitope peptide 10 ⁇ L of PE-labeled MHC for an appropriate amount of PBMC (10 5 to 10 6 cells) or an appropriate amount of CTL line derived using CTL epitope peptide -Tetramer reagent and 20 ⁇ L of FITC-labeled anti-T cell surface antibody (eg CD8, CD4, CD3) etc. were added.
  • FITC-labeled anti-T cell surface antibody eg CD8, CD4, CD3
  • an anti-CD45 antibody labeled with PC5 or the like may be added in order to exclude non-specific fluorescence due to mixed red blood cells. Gently mixed and left at room temperature for 30 minutes. After adding 3 mL of PBS and stirring, it was centrifuged at 400 ⁇ g for 5 minutes.
  • EBV-specific CTL has been difficult with existing methods because the presence of EBV-specific CTL has decreased. If an EBV-specific MHC-tetramer reagent is used, there is a possibility that the presence of EBV-specific CTL can be determined in about 1 hour after blood collection. Therefore, we examined whether EBV-specific CTL could be detected using healthy adult peripheral blood. The result is shown in FIG.
  • peripheral blood of donor ID * 11-11 three types of EBV-specific MHC-tetramer reagent and positive control CMV pp65 MHC-tetramer reagent (MBL) and negative control survivin-2B HLA-A * 24: 02 Stained with MHC-tetramer reagent (MBL).
  • the X-axis is CD8, and the Y-axis is a dot plot development drawing analyzing the fluorescence intensity for the MHC-tetramer reagent on a log scale.
  • the numerical value in the dot plot development diagram represents the ratio of CD8 positive MHC-tetramer reagent positive cells in CD8 positive cells as a positive rate (%).
  • the positive cell population obtained from the negative control HLA-A * 24: 02-restricted survivin-2B MHC-tetramer reagent was 0.17%. It can be said that. From these results, it became clear that EBV LMP2-specific CTLs are present in healthy adult peripheral blood and can grow to such an extent that the presence or absence of CTLs can be determined after 2 weeks of culture. This means that the induction method that does not use antigen-presenting cells, which is carried out in the present invention, is effective for the detection of EBV-specific CTL.
  • ASS (10mer) derived from EBV LMP2 (SEQ ID NO: 1) identified in the present invention has a function of proliferating EBV LMP2-specific CTL in peripheral blood, and these cell populations are ASS (10mer) -Tet.
  • HLA-A * 11: 01-restricted EBV LMP2-specific CTL epitope peptide was found to be an HLA-A * 11: 01-restricted EBV LMP2-specific CTL epitope peptide.
  • the X-axis is CD8
  • the Y-axis is a dot plot developed by analyzing the fluorescence intensity for Tetramer on a log scale.
  • the numerical value in the dot plot development diagram represents the ratio of the MHC-tetramer reagent positive cells in the CD8 positive cells in the living cells as a positive rate (%).
  • the cell population derived from the three mixed peptides (HRG + GVF + KTS) was synthesized using each of the same three peptides and the three mixed MHC-tetramer reagents (HRG) -Tet + GVF-Tet + KTS-Tet), 0.38% positive cells were detected.
  • detection was performed separately using each of the three types of MHC-tetramer reagents. As a result, when stained with HRG-Tet alone, HRG-Tet positive cells were detected with a positive rate of 0.42%.
  • HRG (SEQ ID NO: 26) is an EBV EBNA1-specific CTL epitope peptide showing HLA-A * 11: 01 restriction It has been shown.
  • Intracellular IFN ⁇ production is used as an indicator of the cytotoxic activity of CTL.
  • the inventors performed intracellular IFN ⁇ -producing cell quantification. The result is shown in FIG.
  • the upper part of FIG. 10 is a dot plot development view showing the fluorescence intensity for CD8 on the X-axis and IFN ⁇ on the Y-axis on a log scale. Restimulation with the same peptide as that used for induction was performed to increase the IFN ⁇ production ability. The verified result is shown. The ratio (%) of the number of IFN ⁇ -positive cells in CD8-positive cells to PBMC in UR is shown as a numerical value. The lower part of FIG. 10 shows the result of staining with tetramer when the IFN ⁇ production ability was verified.
  • the X-axis is a dot plot development showing the fluorescence intensity for CD8 on the X-axis and the tetramer on the Y-axis on a log scale.
  • the HRG derived from EBV EBNA1 (SEQ ID NO: 26) identified in the present invention has a function of proliferating EBV EBNA1-specific CTL in peripheral blood, and these cell populations have cytotoxic activity and Since it was detectable with HRG-Tet, it was revealed that it is an HLA-A * 11: 01-restricted EBV EBNA1-specific CTL epitope peptide.
  • the CTL epitope peptide presented on the HLA class I molecule consists of 8 to 10 amino acids, and the 2nd, 9th and 10th amino acids from the N-terminal side are the most important for binding to the HLA class I molecule. It is an amino acid and is called an anchor motif.
  • an anchor motif As a peptide that binds to the HLA-A11 molecule, either Ile, Met, Ser, Thr, or Val is arranged at the second position from the N-terminus, and either Lys or Arg is located at the ninth or tenth position.
  • Peptides consisting of 9 to 10 amino acids are best known (Chang CX, Tan AT, Or MY, Toh KY, Lim PY, Chia AS, Froesig TM, Nadua KD, Oh HL, Leong HN, Hadrup SR, Gehring AJ, Tan YJ, Bertoletti A, Grotenbreg GM.Conditional ligands for Asian HLA variants facilitate the definition of CD8 + T-cell responses in acute and chronic viral diseases.Eur J Immunol. 2013 Apr; 43 (4): 1109-20.
  • the HLA-A * 11: 01-restricted EBV LMP2-specific CTL epitope peptide ASS (10mer) provided by the present invention consists of 10 amino acids, Ala Ser Ser Tyr Ala Ala Ala Gln Arg Lys (SEQ ID NO: 1). It has Ser as the anchor motif, 2nd and 3rd from the N-terminal side, 9th Arg, and 10th Lys. Therefore, for the purpose of clarifying the embodiment of the present invention, a peptide in which one amino acid residue was deleted from the N-terminal side or the C-terminal was synthesized and verified.
  • amino acid sequence of SEQ ID NO: 6 from which N-terminal Ala has been deleted is Ser Ser Tyr Ala Ala Ala Gln Arg Lys
  • amino acid sequence of SEQ ID NO: 5 from which C-terminal Lys has been deleted is Ala Ser Ser Tyr Ala Ala Ala Gln Arg.
  • FIG. 11 a folding test was conducted to examine the ability to form a complex with HLA-A * 11: 01.
  • the MHC-monomer formation rate is ASS (9mer) (337,803 ⁇ V * second), SSY (294,932 ⁇ V * second), ASS (10mer) (238,655 ⁇ V * second) in descending order.
  • the ability to form MHC-monomer was confirmed by comparison with the peptide of the negative control (83,587 ⁇ V * sec).
  • a CTL induction experiment was performed using three types of peptides by the above-described induction method without using antigen-presenting cells. 14 days after induction, specific CTLs were detected using three types of MHC-tetramer reagents.
  • CTL was induced using ASS (10mer), and this was stained using ASS (10mer) -Tet, ASS (9mer) -Tet, and SSY-Tet.
  • ASS (10mer) -Tet positive CD8 positive specific CTL was detected when stained with ASS (10mer) -Tet, but ASS (9mer) -Tet or SSY-Tet positive CD8 positive was detected.
  • ASS (9mer) and SSY peptides have the ability to bind to HLA-A * 11: 01 molecules, they are EBV LMP2-specific CTL precursors ( ASS (10mer) means that there is no ability to stimulate proliferation of effector / memory cells) and CTL induction even if either Ala residue on the N-terminal side or Lys residue on the C-terminal side is deleted And CTL detectability disappeared.
  • IFN ⁇ production ability is detected when restimulated with the same peptide as the peptide used for induction in the cell population induced by CTL using each peptide of ASS (10mer), ASS (9mer), and SSY.
  • ASS 10mer
  • ASS (9mer) ASS
  • SSY SSY
  • 40.52% of ATV-Tet positive cells are present in the cell population induced using HLA-A * 11: 01 restricted epitope peptide (ATVQGQNLK, SEQ ID NO: 34) derived from CMV pp65 protein, which is a peptide for positive control, 28% of cells produced IFN ⁇ upon restimulation with ATV peptide.
  • survivin-2B HLA-A * 24: 02 restricted epitope peptide (AYACNTSTL, SEQ ID NO: 35) used as a negative control peptide 0.17% of AYA-Tet positive cells were detected. The cell population is not detected as a clear spot and is considered to be a non-specific reaction.
  • 8.67% of IFN ⁇ -positive cells were detected by restimulation with AYA peptide, which is a level of non-specific staining that is common in intracellular IFN ⁇ staining.
  • the cell population induced by using the EBV LMP2-derived HLA-A * 11: 01-restricted ASS (10mer) identified in the present invention has 53.31% ASS (10mer) -Tet-specific CTL, and the ASS (10mer) 68% of the cell population produced IFN ⁇ by restimulation.
  • ASS in the case of SSY lacking the N-terminal Ala residue, no SSY-Tet positive cells were detected, and IFN ⁇ production was also lower than that of the negative control. From this, even if it is a peptide having an amino acid corresponding to an anchor motif and also forms an MHC-monomer, it cannot always specifically induce CTL as an epitope peptide. It has been shown that an epitope peptide capable of inducing CTL cannot be easily conceived.
  • This solution includes a method using a plurality of CTL epitopes for one type of HLA and a method using each CTL epitope for a plurality of HLA types. is there. For example, when the subject patient holds HLA-A2 and HLA-A11, it is desirable that a plurality of HLA-restricted CTL epitopes are selected for treatment. This is one of the problems to be solved by the present invention.
  • the results are shown in FIG.
  • the new epitope ASS (10mer) is GD2 (from Guangdong, China) and HKNPC1 (from a Hong Kong NPC patient), with the second and fourth Tyr residues mutated to Asn and Ser residues, respectively. (S2N Y4S) was found.
  • the novel epitope HRG was found to be mutated in two strains.
  • EBV genomic DNA was extracted from PBMC of 5 donors or B95.8 cell line (ATCC) using GeneJET Viral DNA and RNA Purification Kit (Thermo scientific). Using this as a template and the above primers, the Exon 2 fragment was amplified by PCR reaction and cloned using TOPO (registered trademark) TA cloning Kit (Life technologies). Three clones were selected from each specimen and sequence analysis was performed. The results of the sequence are shown in FIG. In FIG.
  • ASSYAAAQRK (base sequence: GCCAGCTCATATGCCGCTGCACAAAGGAAA (SEQ ID NO: 48), GCCAGCTCATATGCCGCTGCACAGAGGAAA (SEQ ID NO: 49)) observed between EBV strains are shown in close-up.
  • Table 6 shows the ASS (10mer) sequence results. Compared to B95.8, donor * 11-11 in the DNA sequence, but was seen (CA G from CAA) single base substitution was not an amino acid substitution. The other 4 individuals had the same sequence as B95.8. From these results, five Japanese donors used in the present invention were infected with the most widely distributed B95.8 strain, and mutations in the ASS (10mer) CTL epitope could not be detected.
  • HLA-A * 11 01-restricted EBV LMP2 ASS (10mer) -specific CTL was detected by ANS (S2N) -Tet as shown in FIG. 17, ANS (S2N) -Tet was detected by ASS (10mer ) -Tet cross-reactivity was maintained (26.87% vs. 27.63%), but ASS (Y4S) -Tet could hardly detect positive cells and lost its binding to TCR ( 0.49%). ASN (S2N, Y4S) -Tet retained some ability to bind to TCR (5.93%).
  • this epitope is functionally complementary, that is, it is possible to avoid a decrease in immune response due to amino acid mutation caused by the difference between GD2 strain and HKNPC1 strain and other infected EBV strains when both are used in combination.
  • novel epitope HRG derived from HLA-A * 11: 01-restricted EBV EBNA1 includes HRG derived from AG876 strain (Q4E, N8S, P9Q) and HRG derived from Mutu strain (P9Q).
  • HRG derived from AG876 strain Q4E, N8S, P9Q
  • HRG derived from Mutu strain P9Q
  • the present invention has succeeded in identifying a novel CTL epitope specific to EBV LMP2 or EBV EBNA1, and can realize antigen-specific cytotoxic T cell therapy (CTL therapy) targeting HLA-A11 carriers. It was. CTL therapy is expected as a next generation immunotherapy.
  • CTL therapy is expected as a next generation immunotherapy.
  • CPC medical grade cultured cell processing center
  • FIG. 18 shows the result of observing cells during the culture period with a microscope. PBMCs stimulated with anti-CD3 antibody were observed to activate T cell masses on day 7 and PBMCs stimulated with peptide on day 14; one week after starting mixed culture (day 21), activated T cell masses Further growth was shown.
  • FIG. 19 shows the total number of cells during the culture period and the number of CTLs proliferated by induction of four types of peptides.
  • the CTL number was calculated from the tetramer staining result of FIG.
  • FIG. 19 is a dot plot development view showing the fluorescence intensity for CD8 on the X axis and the tetramer on the Y axis on a log scale, and was stained with a tetramer of the same peptide as that used for induction.
  • the numerical value in the dot plot development diagram represents the ratio of the MHC-tetramer reagent positive cells in the CD8 positive cells in the living cells as a positive rate (%).
  • the above culture system examined by the inventors is characterized in that it is performed in a closed system instead of a conventional open system. Therefore, it is considered that operability, economy and safety in preparation of antigen-specific CTL can be realized, and the practical use of CTL therapy can be promoted.
  • Each peptide of SEQ ID NO: 1 was dissolved in DMSO to a final concentration of 20 mg / ml, and sterilized by filtration. The obtained peptide-containing solution was dispensed and sealed in 1 mL sterilized vials to obtain a vaccine injection.
  • CKAP4 is a type II membrane protein composed of 602 amino acids in total length, and no isoform has been reported. Selection of CKAP4-specific CTL epitope candidate peptides of the present invention was performed against HLA-A * 24: 02 possessed by about 60% of Japanese. Specifically, we collated with multiple software published on the Internet that can search for CTL epitope candidate peptides consisting of 8 to 12 amino acids having a binding motif for HLA-A * 24: 02. Carried out.
  • CTL epitope candidate peptides consisting of 9 to 10 amino acids having a binding motif of HLA-A * 24: 02 were selected from the amino acid sequence of CKAP4 and synthesized.
  • the synthesized CTL epitope candidate peptides are shown below.
  • Table 7 shows the characteristics of the synthesized HLA-A * 24: 02-binding CKAP4-specific CTL epitope candidate peptides.
  • Three or four amino acid sequences from the N-terminal side of the synthesized CKAP4-specific CTL epitope candidate peptide are abbreviated as peptide names. From left, peptide name, amino acid sequence, position on CKAP4 amino acid sequence, number of amino acids, HLASPeptide of BIMAS (BioInformatics & Molecular Analysis Section / http://thr.cit.nih.gov/index.shtml) used for analysis The score calculated by Binding Predictions (http://thr.cit.nih.gov/molbio/hla_bind/) is shown.
  • This score is a numerical value that predicts the affinity between HLA-A * 24: 02 and the peptide. The higher the score, the more likely the HLA molecule and peptide may form a stable complex.
  • analysis software BIMAS described in Table 7 the inventors selected 10 types (SEQ ID NOs: 36 to 45) of CTL epitope candidate peptides using analysis software such as SYFPEITHI, Rankpep, IEDB Bind prediction, and NetCTL.
  • [Folding test of CKAP4-specific CTL epitope candidate peptide] The inventor conducted a folding test using ten types of artificially synthesized CKAP4-specific CTL epitope candidate peptides. Specifically, HLA-A * 24: 02 expressed and purified using an E. coli expression system, ⁇ 2-microglobulin, and CKAP4-specific CTL epitope candidate peptide are added to the folding solution, mixed, and then folded over time. The solution was collected and analyzed with a gel filtration column.
  • MHC-monomer When gel filtration column analysis shows formation of a ternary complex (MHC-monomer) of HLA-A * 24: 02, ⁇ 2-microglobulin, and CKAP4-specific CTL epitope candidate peptide, MHC-monomer Since the molecular weight is large, the elution time in the gel filtration column analysis is shortened. Further, the amount of MHC-monomer formation can be calculated from the peak area obtained by the absorption wavelength of 280 nm. On the other hand, MHC-monomer formation is not confirmed with candidate peptides that do not bind to HLA molecules. A typical gel filtration column analysis example in which MHC-monomer formation is observed is shown in FIG.
  • HLA molecules and ⁇ 2-microglobulin are purified as inclusion bodies, which are insoluble fractions, and are solubilized with 8M urea when purified using the E. coli expression system.
  • HLA molecules that are hardly soluble those that do not lead to MHC-monomer formation are detected as aggregates in 7 to 8 minutes. However, most of the aggregates are removed by filter filtration before gel filtration column analysis.
  • ⁇ 2-microglobulin is a soluble protein that is solubilized in the folding solution and detected around 14 minutes. After 15 minutes, the folding solution composition and peptide are detected.
  • the peak of MHC-monomer is not confirmed immediately after the start of the folding test (day 0), but the peak increases after 1 day (day 1) and 3 days (day (3), and MHC-monomer formation is progressing smoothly. Shows things.
  • FIG. 22 shows the analysis results after 4 days of the folding test performed on 10 kinds of CKAP4-specific CTL epitope candidate peptides.
  • a positive control peptide consisting of 9 amino acids SEQ ID NO: 46
  • a negative control SEQ ID NO: 47
  • Control peptide used for folding test Ala Tyr Ala Cys Asn Thr Ser Thr Leu (SEQ ID NO: 46) Ser Ser Tyr Arg Arg Pro Val Gly Ile (SEQ ID NO: 47)
  • the graph shows the peak area indicating MHC-monomer formation as a bar graph.
  • CKAP4-specific MHC-tetramer reagent [Production of CKAP4-specific MHC-tetramer reagent] Based on the results of the folding test, PE (phycoerythrin) -labeled MHC-tetramer using CKAP4-specific CTL epitope candidate peptides of SEQ ID NOs: 36, 40, 42 and 43, HLA-A * 24: 02, and ⁇ 2-microglobulin Reagents were manufactured.
  • the MHC-tetramer reagent produced in the present invention is abbreviated as, for example, KVQE-Tet. It shows what was manufactured using the person complex.
  • MHC-monomer a complex of HLA class I molecule purified from a recombinant host for protein expression, ⁇ 2-microglobulin, and a CKAP4-specific CTL epitope candidate peptide of the present invention is formed in an appropriate folding solution.
  • a biotin binding site is added to the C-terminus of the recombinant HLA class I molecule in advance, and biotin is added to this site after MHC-monomer formation.
  • a MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4.
  • HLA-A * 24 It is desirable to verify using the peripheral blood of the donor who holds: 02. First, whether or not a donor has HLA-A * 24: 02 was confirmed by genotyping of HLA-A using Genosearch (trademark) HLA-A Ver.2 (MBL). Subsequent examinations were performed using PBMCs of 7 healthy adults with HLA-A * 24: 02.
  • MLPC Mated Lymphocyte-Peptide Cultures
  • the MLPC method is a method of inducing CTL by adding a peptide to a PBMC culture solution (Karanikas V, Lurquin C, Colau D, van Baren N, De Smet C, Lethe B, Connerotte T, Corbiere V, Demoitie MA , Lienard D, Dreno B, Velu T, Boon T, Coulie PG.Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus.J Immunol.
  • Peripheral blood collected from healthy adults carrying HLA-A * 24: 02 was centrifuged at 3,000 rpm for 5-10 minutes, and the plasma portion of the supernatant was collected. Except for the plasma portion, PBMCs were separated by the conventional density gradient centrifugation method. In the present invention, good results were obtained using a medium supplemented with 5% plasma. The medium is generally supplemented with appropriate additives and antibiotics for the medium used for cell culture.
  • the CTL induction medium used in the present invention a medium in which RPMI1640 Hepes modify (Sigam) was added with 2-mercaptoethanol, L-glutamine, and streptomycin and penicillin as antibiotics was used.
  • PBMCs peripheral blood mononuclear cells
  • CKAP4-specific CTL epitope candidate peptides SEQ ID NOs: 36, 40, 42, and 43
  • concentration of the peptide can be varied depending on the solubility of the peptide. In the present invention, it was carried out at 10 ⁇ g / mL.
  • a 96-well U-bottom cell culture microtest plate (BECTON DICKINSON) was used. The cells were cultured in a 37 ° C., 5% CO 2 constant temperature bath. Two days later, IL-2 was added at a final concentration of 20-100 U / mL. Thereafter, the IL-2 added CTL induction medium was appropriately replaced. Confirmation of CKAP4-specific CTL was carried out within 2 weeks of culture. When induction of CKAP4-specific CTL was confirmed, stimulation with peptide pulse antigen-presenting cells or direct stimulation with a peptide was attempted to establish a CTL line.
  • CKAP4-specific CTL Whether or not CKAP4-specific CTL is present in the cell population cultured by the above-described method was examined by the MHC-tetramer method. After culturing, 10 ⁇ L of PE-labeled MHC-tetramer reagent, 20 ⁇ L of FITC (fluorescein isothiocyanate) -labeled T cell surface antibody (eg, CD8, CD4, CD3) and the like were added to an appropriate amount of cells. Furthermore, a CD45 antibody labeled with PC5 (phycoerythrin-Cy5) or the like may be added in order to exclude non-specific fluorescence due to contaminated erythrocytes.
  • FITC fluorescein isothiocyanate
  • the mixture was gently mixed and allowed to stand at 2-8 ° C. for 60 minutes or at room temperature for 30 minutes. After adding 1.5 mL of PBS and stirring, the mixture was centrifuged at 3,000 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 400 ⁇ L of PBS. At this time, 7-AAD viability dye (dead cell detection reagent, MBL) may be added in order to exclude non-specific fluorescence due to dead cells. The analysis was performed with a flow cytometer within 24 hours.
  • 7-AAD viability dye dead cell detection reagent, MBL
  • the first step is to collect some of the cells from each of the 8 wells in a 96-well U-bottom cell culture microtest plate and pool them as one sample (lane pool) to check for the induction of CKAP4-specific CTL. This was confirmed by the MHC-tetramer method.
  • the second stage cells in each well were individually collected in the lane pool in which the induction of CKAP4-specific CTL was confirmed in the first stage, and the presence or absence of induction of CKAP4-specific CTL was confirmed by the MHC-tetramer method. By using such a method, it was confirmed in which well of the 96-well U-bottom cell culture microtest plate that CKAP4-specific CTL was induced.
  • FIG. 23 and FIG. 25 show typical results of confirmation at the first stage after induction of CKAP4-specific CTL by MLPC method.
  • PBMCs of specimen numbers A24-37 (FIG. 23) and A24-39 (FIG. 25) were cultured with SEQ ID NO: 43 of a CKAP4-specific CTL epitope candidate peptide for 14 days.
  • the numbers in the dot plot development diagram are (UR + LR) for the area obtained by dividing the development quadrant into UL (upper left), UR (upper right), LL (lower left), and LR (lower right). Shows the percentage of UR.
  • the X-axis is CD8, and the Y-axis is a dot plot development view showing the fluorescence intensity for the MHC-tetramer reagent on a log scale.
  • IYT-Tet confirmed the induction of the specific CTL of SEQ ID NO: 43.
  • Species number A24-37 was lane 7, A24-39 was lane 11 UR, CD8 positive IYT-Tet positive cells A population was detected. This indicates that SEQ ID NO: 43 is a CKAP4-specific CTL epitope peptide and memory type CKAP4-specific CTL was present in the peripheral blood of specimens A24-37 and A24-39.
  • FIG. 26 show the results of the second step in which the CTL specific for SEQ ID NO: 43 was induced by MLPC method and detected by MHC-tetramer method.
  • FIG. 23 and FIG. 25 show the results of confirming the presence or absence of induction of CKAP4-specific CTL in each well by MHC-tetramer method in each lane where the induction of CKAP4-specific CTL was confirmed.
  • the sample number A24-37 lane 7 G well (7-G) shown in FIG. 24 and the sample number A24-39 lane 11 B well (11-B) shown in FIG. CTL was detected.
  • SEQ ID NO: 43 is a CKAP4-specific CTL epitope peptide exhibiting HLA-A * 24: 02 restriction.
  • SEQ ID NO: 43-specific CTL was detected in 1 of 96 wells, respectively. Therefore, the abundance ratio of SEQ ID NO: 43-specific CTL in peripheral blood PBMC was It is calculated by the following formula.
  • the limit of detection of specific CTLs by the 96-well MLPC method is usually 1.6 to 3.3 ⁇ 10 ⁇ 7 assuming that the CD8 positive cell rate before induction is 10 to 20%.
  • the standard ratio of specific CTL detected is 1 per 3-6 ⁇ 10 6 CD8 positive cells.
  • the present invention was carried out in the same manner for 7 blood donors using SEQ ID NOs: 36, 40, 42, and 43 as shown in Table 8, but specific CTLs were detected except for SEQ ID NO: 43. I could't. Even with SEQ ID NO: 43, only 2 out of 7 were detected. This indicates that it is difficult to predict a CKAP4-specific HLA-A * 24: 02-restricted epitope peptide, and it is more difficult to predict whether the epitope peptide has the ability to induce specific CTLs. ing.
  • EBV-infected B cell lines Kuzushima K, Yamamoto M, Kimura H, Ando Y, Kudo T, Tsuge I, Morishima T.
  • EBV Epstein-Barr virus
  • EBV-infected LCL EBV-infected B cell line
  • [Amplification of specific CTL using antigen-presenting cells] Suspend the antigen-presenting cells (EBV-infected LCL) in pulse medium (0.5% human serum albumin / RPMI1640) or AIM-V medium (Invitrogen), and add CTL epitope candidate peptide at a concentration of 10 ⁇ g / mL The mixture was allowed to stand at room temperature for 30 to 60 minutes with gentle mixing at intervals of approximately 15 minutes, and then washed three times with an excessive amount of pulse medium to wash away unbound peptides to HLA molecules. By performing this operation, it is considered that the CTL epitope candidate peptide binds to the HLA molecule on the antigen-presenting cell.
  • the antigen-presenting cell subjected to this operation is called a peptide pulse antigen-presenting cell.
  • Peptide pulse antigen-presenting cells were treated with a lethal dose of X-rays or mitomycin to lose their ability to grow. This was mixed with a cell population containing CKAP4-specific CTL derived from the same person and cultured in a 37 ° C., 5% CO 2 constant temperature bath.
  • RPMI1640 medium containing 10% fetal calf serum (FCS), RPMI1640 medium containing 10% human serum, or RPMI1640 medium containing 1-10% human plasma was examined. In this method, Good results were obtained with RPMI1640 medium containing 5% human plasma.
  • IL-2 (Shionogi Pharmaceutical Co., Ltd.) was added at 50 U / mL for the purpose of maintaining the survival of T cells and assisting proliferation. Evaluation of CKAP4-specific CTL induction was performed 10 to 16 days after the start of the culture. When induction of CKAP4-specific CTL was confirmed, stimulation was further performed using peptide pulse antigen-presenting cells to establish a CTL line.
  • FITC-labeled anti-IFN ⁇ antibody manufactured by MBL was added and allowed to react at room temperature for 15-30 minutes. After washing, the IFN ⁇ positive cell rate in T cells or the IFN ⁇ positive cell rate in MHC-tetramer reagent positive cells was quantified using a flow cytometer.
  • FIG. 27 shows the results of examination by an intracellular IFN ⁇ producing cell quantification method of CKAP4-specific CTL induced.
  • PBMC of specimen number A24-39 was stimulated with SEQ ID NO: 43, which is an HLA-A * 24: 02-restricted CKAP4-specific CTL epitope peptide.
  • SEQ ID NO: 43 which is an HLA-A * 24: 02-restricted CKAP4-specific CTL epitope peptide.
  • MBL PE-labeled MHC-tetramer reagent
  • PC5-labeled CD8 antibody PC5-labeled CD8 antibody
  • FITC-labeled IFN ⁇ antibody PE-labeled IFN ⁇ antibody
  • IFN ⁇ -positive MHC-tetramer reagent-positive cells appear in UR only when stimulated with a specific peptide, and hardly appear when no peptide is added.
  • the presence of IYT-Tet-specific CTLs in both the cell population with and without the peptide indicates that the fluorescence intensity for CD8 on the X axis and the MHC-tetramer reagent on the Y axis is log. It is clear from the dot plot development shown on the scale that the CD8 positive MHC-tetramer reagent positive cell population is seen in the UR.
  • CKAP4-specific CTL having cytotoxic activity to produce IFN ⁇ was induced by restimulation in PBMC cultured with CKAP4-specific CTL epitope peptide, and these cells were stained with MHC-tetramer reagent. From this, it was revealed that the CTL was specific to the HLA-A * 24: 02-restricted CKAP4-derived peptide IYTEVRELV (SEQ ID NO: 43).
  • the present invention makes it possible to treat or prevent EBV infection and a virus-positive cancer using a cytotoxic T cell epitope peptide specific to Epstein-Barr virus.
  • CTL specific for EBV can be quantified.
  • HLA-A * 24: 02-restricted epitope peptide comprising the sequence of IYTEVRELV (SEQ ID NO: 43) of the present invention it becomes possible to treat diseases dependent on malignant tumor cells that highly express CKAP4. .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un épitope peptidique de cellules T cytotoxiques (lymphocytes T cytotoxiques, ci-après abrégés sous la forme CTL, pour "cytotoxic T lymphocyte") spécifique du virus d'Epstein-Barr (ci-après décrits sous la forme EBV, pour "Epstein-Barr virus"), un vaccin pour traiter ou prévenir une infection à EBV ou un cancer positif pour l'EBV par utilisation de ce peptide, un agent d'immunothérapie passive contre l'EBV, et une méthode d'analyse des CTL spécifiques de l'EBV. La présente invention concerne également un épitope peptidique restreint au HLA-A*24:02 comprenant une séquence IYTEVRELV (SEQ ID NO : 43) issue d'une protéine du cytosquelette (protéine associée au cytosquelette de type 4 : ci-après nommée CKAP4, également connue sous les noms : CLIMP-63, ERGIC-63, P63). Les cellules T cytotoxiques spécifiques du peptide (ci-après nommées CTL) peuvent attaquer les cellules des tumeurs malignes qui expriment un niveau élevé de CKAP4.
PCT/JP2015/067469 2015-06-17 2015-06-17 Épitope peptidique de cellules t cytotoxiques et utilisation correspondante WO2016203577A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2017524207A JPWO2016203577A1 (ja) 2015-06-17 2015-06-17 細胞傷害性ｔ細胞エピトープペプチド及びその用途
CN201580080723.XA CN107709351A (zh) 2015-06-17 2015-06-17 细胞毒性t细胞表位肽及其用途
PCT/JP2015/067469 WO2016203577A1 (fr) 2015-06-17 2015-06-17 Épitope peptidique de cellules t cytotoxiques et utilisation correspondante
TW105119067A TW201708248A (zh) 2015-06-17 2016-06-17 細胞毒性ｔ淋巴球抗原決定位胜肽及其用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2015/067469 WO2016203577A1 (fr) 2015-06-17 2015-06-17 Épitope peptidique de cellules t cytotoxiques et utilisation correspondante

Publications (1)

Publication Number Publication Date
WO2016203577A1 true WO2016203577A1 (fr) 2016-12-22

Family

ID=57545291

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/067469 WO2016203577A1 (fr) 2015-06-17 2015-06-17 Épitope peptidique de cellules t cytotoxiques et utilisation correspondante

Country Status (4)

Country Link
JP (1) JPWO2016203577A1 (fr)
CN (1) CN107709351A (fr)
TW (1) TW201708248A (fr)
WO (1) WO2016203577A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108997481A (zh) * 2017-06-07 2018-12-14 中国科学院广州生物医药与健康研究院 源自于lmp1的抗原短肽
KR20190017705A (ko) * 2017-08-10 2019-02-20 주식회사 굳티셀 암 치료를 위한 t 세포의 활성화 방법
WO2019054409A1 (fr) * 2017-09-12 2019-03-21 国立大学法人北海道大学 Procédé de criblage et kit de criblage d'une substance interagissant avec des protéines hla
CN113597430A (zh) * 2019-02-08 2021-11-02 古德T细胞有限公司 激活t细胞用于癌症治疗的方法
WO2021232048A1 (fr) * 2020-05-11 2021-11-18 Board Of Regents, The University Of Texas System Cellules t spécifiques au sars-cov-2

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109294983A (zh) * 2018-09-30 2019-02-01 北京鼎成肽源生物技术有限公司 一种lff2细胞
CN110007071A (zh) * 2019-04-01 2019-07-12 河南省生物工程技术研究中心有限公司 一种ckap4标记抗体-荧光微球复合物的制备方法及其应用
CN110054663B (zh) * 2019-04-01 2023-10-03 河南省生物工程技术研究中心 Ckap4抗原表位肽、抗原、单克隆抗体及其应用和ckap4检测试纸条
CN110638787B (zh) * 2019-07-31 2021-11-05 中山大学 一种防治鼻咽癌的亚单位纳米疫苗及其制备方法
CN113419067A (zh) * 2021-05-26 2021-09-21 广州医科大学 一种用于评估造血干细胞移植病人cmv感染后免疫状态的多肽组合物以及检测试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007117023A (ja) * 2005-10-28 2007-05-17 Igaku Seibutsugaku Kenkyusho:Kk エプスタイン−バールウイルス感染細胞を特異的に攻撃する細胞傷害性ｔ細胞エピトープペプチド及びその用途
WO2009105152A2 (fr) * 2008-01-31 2009-08-27 University Of Massachusetts Medical School Particules pseudo-virales servant de vaccins pour le paramyxovirus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007117023A (ja) * 2005-10-28 2007-05-17 Igaku Seibutsugaku Kenkyusho:Kk エプスタイン−バールウイルス感染細胞を特異的に攻撃する細胞傷害性ｔ細胞エピトープペプチド及びその用途
WO2009105152A2 (fr) * 2008-01-31 2009-08-27 University Of Massachusetts Medical School Particules pseudo-virales servant de vaccins pour le paramyxovirus

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BAO Y. N. ET AL.: "Integrating transcriptome profiling and in silico pathway analysis toward reduction of therapeutic dimensionality in nasopharyngeal carcinoma", CANCER RES., vol. 71, no. 8, 2011 *
CHEUNG W. H. ET AL.: "Conjugation of latent membrane protein (LMP)-2 epitope to gold nanoparticles as highly immunogenic multiple antigenic peptides for induction of Epstein- Barr virus-specific cytotoxic T-lymphocyte responses in vitro", BIOCONJUG. CHEM., vol. 20, no. 1, 2009, pages 24 - 31 *
LI S. X. ET AL.: "Prognostic significance of cytoskeleton-associated membrane protein 4 and its palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma.", CANCER, vol. 120, no. 10, 2014, pages 1520 - 31 *
MURRAY R. J. ET AL.: "Epstein-Barr virus- specific cytotoxic T- cell recognition of transfectants expressing the virus-coded latent membrane protein LMP", J. VIROL., vol. 62, no. 10, 1988, pages 3747 - 55 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108997481A (zh) * 2017-06-07 2018-12-14 中国科学院广州生物医药与健康研究院 源自于lmp1的抗原短肽
CN108997481B (zh) * 2017-06-07 2022-06-03 中国科学院广州生物医药与健康研究院 源自于lmp1的抗原短肽
WO2019031939A3 (fr) * 2017-08-10 2019-07-18 주식회사 굳티셀 Méthode d'activation des cellules t pour le traitement du cancer
CN110997903A (zh) * 2017-08-10 2020-04-10 古德T细胞有限公司 用于癌症治疗的t细胞的活化方法
CN111433355A (zh) * 2017-08-10 2020-07-17 古德T细胞有限公司 用于癌症治疗的t细胞的活化方法
KR102148866B1 (ko) 2017-08-10 2020-10-14 주식회사 굳티셀 암 치료를 위한 t 세포의 활성화 방법
KR20190017705A (ko) * 2017-08-10 2019-02-20 주식회사 굳티셀 암 치료를 위한 t 세포의 활성화 방법
US11918634B2 (en) 2017-08-10 2024-03-05 Good T Cells, Inc. Method for activating T cells for cancer treatment
CN111433355B (zh) * 2017-08-10 2024-03-29 古德T细胞有限公司 用于癌症治疗的t细胞的活化方法
CN110997903B (zh) * 2017-08-10 2024-03-29 古德T细胞有限公司 用于癌症治疗的t细胞的活化方法
US11969463B2 (en) 2017-08-10 2024-04-30 Good T Cells, Inc. Method for activating T cells for cancer treatment
WO2019054409A1 (fr) * 2017-09-12 2019-03-21 国立大学法人北海道大学 Procédé de criblage et kit de criblage d'une substance interagissant avec des protéines hla
CN113597430A (zh) * 2019-02-08 2021-11-02 古德T细胞有限公司 激活t细胞用于癌症治疗的方法
WO2021232048A1 (fr) * 2020-05-11 2021-11-18 Board Of Regents, The University Of Texas System Cellules t spécifiques au sars-cov-2

Also Published As

Publication number Publication date
JPWO2016203577A1 (ja) 2018-04-12
CN107709351A (zh) 2018-02-16
TW201708248A (zh) 2017-03-01

Similar Documents

Publication Publication Date Title
WO2016203577A1 (fr) Épitope peptidique de cellules t cytotoxiques et utilisation correspondante
US11707512B2 (en) Cancer vaccine composition
JP4824389B2 (ja) エプスタイン−バールウイルス感染細胞を特異的に攻撃する細胞傷害性ｔ細胞エピトープペプチド及びその用途
AU2015343239B2 (en) Methods of selecting T cell line and donor thereof for adoptive cellular therapy
JP4976294B2 (ja) 細胞傷害性ｔ細胞エピトープペプチド及びその用途
CA2777821A1 (fr) Peptides associes a une tumeur qui se lient aux molecules mhc
Kuzushima et al. Tetramer-assisted identification and characterization of epitopes recognized by HLA A* 2402–restricted Epstein-Barr virus–specific CD8+ T cells
CA2431500A1 (fr) Peptides longs de 22-45 residus d'acides amines induisant et/ou stimulant les reponses immunitaires specifiques de l'antigene
US20130217122A1 (en) Expansion of Interferon-Gamma-Producing T-Cells Using Glypican-3 Peptide Library
TWI374031B (fr)
Boyle et al. Major histocompatibility complex class I‐restricted alloreactive CD4+ T cells
JP2019110909A (ja) 細胞傷害性ｔ細胞エピトープペプチド及びその用途
WO2017086354A1 (fr) Peptide d'épitope de cellule t cytotoxique à restriction ｈｌａ－ａ１１
Zentz et al. Activated B cells mediate efficient expansion of rare antigen-specific T cells
Provenzano et al. The matrix protein pp65341‐350: a peptide that induces ex vivo stimulation and in vitro expansion of CMV‐specific CD8+ T cells in subjects bearing either HLA‐A* 2402 or A* 0101 allele
WO2022244891A1 (fr) PEPTIDE D'ÉPITOPE DE LYMPHOCYTE T DÉRIVÉ DU SARS-CoV-2
JP2017132745A (ja) Ｈｈｖ−６ｂ特異抗原ｕ５４由来ｈｌａ−ａ２４拘束性ｃｔｌエピトープペプチド及びその使用
Meckiff Evolution of the human CD4+ T cell response to Epstein-Barr virus infection-analysis of systemic and local immune responses
JP2011239787A (ja) エプスタイン−バールウイルス感染細胞を特異的に攻撃する細胞傷害性ｔ細胞エピトープペプチド及びその用途
Preece Harnessing CD4+ T cell effectors for lymphoma therapy
ZHENYING Amino acid substitutions in the epstein-barr virus associated oncogene latent membrane protein 1 impact upon the immunogenicity of Nasopharyngeal carcinoma cells
Provenzano et al. TRANSPLANTATION AND CELLULAR ENGINEERING

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15895598

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017524207

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15895598

Country of ref document: EP

Kind code of ref document: A1