JP2019110909A - Epitope peptides of cytotoxic t lymphocyte and uses thereof - Google Patents

Abstract

Kind Code: A1 A vaccine for treating or preventing Epstein-Barr virus (Epstein-Barr virus, hereinafter referred to as EBV) infection and virus-positive cancer, a passive immunotherapy against EBV, and quantification of EBV-specific CTLs. provide a way. A cytotoxic T lymphocyte (CTL) epitope peptide specific to EBV. An HLA-A*24:02-restricted epitope peptide derived from cytoskeleton-associated protein 4 (hereinafter referred to as CKAP4, also known as CLIMP-63, ERGIC-63, P63) and consisting of the IYTEVRELV sequence. The peptide-specific cytotoxic T cells can attack malignant tumor cells that highly express CKAP4. [Selection figure] None

Description

  The present invention relates to an epitope peptide of cytotoxic T cells (hereinafter referred to as CTL) epitope peptide specific to Epstein-Barr virus (hereinafter referred to as "Epstein-Barr virus", hereinafter referred to as "EBV"), an epitope peptide of EBV using said peptide. The present invention relates to a vaccine for treating or preventing an infection and a virus positive cancer, a passive immunotherapeutic agent for EBV, and a method for quantifying EBV-specific CTL. Furthermore, the present invention relates to a peptide capable of inducing CTL that targets cancer cells. The present invention also relates to a cancer vaccine and an anticancer agent comprising the peptide. Furthermore, the present invention relates to the use of the peptide for inducing CTL targeting cancer cells, the obtained CTL and an anticancer agent comprising the CTL.

  In 1964, Epstein and Barr discovered a new herpesvirus from cultured cells derived from Burkitt's lymphoma tissue by Epstein and Barr, 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 herpesvirus (HHV), and is a virus which is latently infected widely worldwide (Non-patent Document 2). An infection is usually established in the oral and pharyngeal mucosa via saliva in childhood, and the virus produced by the oral and pharyngeal mucosal epithelial cells is further infected by B cells passing between the epithelia and spreads throughout the body. The immune surveillance mechanism, mainly by cytotoxic T cells (CTL), damages and kills infected B cells, but some become latent infections that do not produce virus.

  The discovery of DNA fragments derived from EBV virus from nasopharyngeal cancer tissues in 1970 made EBV the first tumor virus among viruses that infect humans (Non-patent Documents 3 and 4) . In addition to EBV, typical examples of tumor virus 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. T cell leukemia virus (HTLV-1) and the like.

  EBV mainly infects B cells, which are one of human lymphocyte components, but also infects epithelial cells, T cells, NK cells, etc. in addition to B cells. It is believed to be involved with For example, cancers believed to be caused by EBV infection include malignant tumors such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), NK / T cell lymphoma, nasopharyngeal cancer (NPC), gastric cancer (MK) and the like. Be EBV is also deeply involved in multiple immunodeficiency diseases, including, for example, lymphoproliferative disease (LPD), infectious mononucleosis (IM), posttransplant lymphoproliferative disease (PTLD), etc. (Non-Patent Document 4).

  Recent studies have revealed that EBV is also involved in autoimmune diseases. In patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome (SS), etc., an increase in the viral load of EBV has been reported. In addition, EBV-derived small RNA (EBV encoded small RNA, EBER) and several proteins are thought to be deeply related to the onset and progression of autoimmune diseases, and their mechanisms have been elucidated. (Non-Patent Documents 5, 6, 7).

  Thus, since EBV is considered to be closely related to cancer and immunodeficiency diseases, clinical research on immune cell therapy targeting EBV has greatly progressed in the past ten years, and remarkable results have been achieved. It is contained. For example, so-called immune cells are transfused into patients with immunocompetent cells that can eliminate causative virus-infected cells against EBV-related lymphoproliferative disease (LPD), posttransplant lymphoproliferative disease (PTLD) The therapy was performed, and its therapeutic effect was proved (Non-patent Documents 8, 9, 10). In addition, the efficacy of immune cell therapy has also been reported for EBV-related malignancies such as Hodgkin's lymphoma and nasopharyngeal cancer (Non-Patent Documents 11, 12, 13, 14, 15).

  Because of their effective clinical research, immune cell therapy is of great interest as a new cancer therapeutic strategy. In conventional surgery, radiation therapy, chemotherapy and refractory malignancies and immunodeficiency diseases, immune cell therapy is considered to be a potential treatment.

  Immune cell therapy is a therapeutic method using activation of the patient's immune system and specific attack / elimination of target cancer cells or virus-infected cells. is there. Cytotoxic T cells (CTLs) play a central role in specifically attacking these target cells. When CTL recognizes a target cell such as a cancer cell or a virus-infected cell, a T cell receptor (TCR) expressed on the CTL cell membrane surface plays an important role. The TCR does not directly recognize the cancer antigen molecule or the virus particle itself, but the HLA (human leukocyte type antigen) expressed on the membrane surface of the target cell and the tumor antigen derived from the cancer antigen or virus infection In the case of cells, CTLs recognize target cells and exert 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 roughly divided into class I and class II, complexes of HLA class I and peptide are recognized by TCRs expressed on CD8 positive T cells, complexes of HLA class II and peptides are expressed on CD4 positive T cells Are recognized to elicit an immune response. The 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. The HLA compatibility of donors and recipients in transplantation medical care is important for the six-locus compatibility of HLA-DRB classified into HLA-A, B and HLA class II, especially in transplantation among unrelated persons. It is considered as a risk factor in According to IMGT's HLA database (http://www.imgt.org/), 2,041 proteins for HLA-A, 2,688 types for HLA-B, and 1,677 types for HLA-C are registered. However, it is known that there is a bias among races, for example, about 60% of Japanese people hold HLA-A24, HLA-A2 is 50% or more of white people, and Japanese people also have about 20 % Is holding. The EBV-derived LMP2 and EBNA1 specific CTL epitope peptides identified by the inventors specifically bind to HLA-A11, and the CTLs that recognize it specifically kill and eliminate EBV-infected cells. The possession rate of HLA-A11 is about 10% in Japanese, but in Southeast Asia, it has a broad population distribution as the third largest possession allyl. Allele frequencies of 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).

  Most of the immune cell therapies that have been reported so far have the largest number of clinical trials for HLA-A2 worldwide, and in Japan there are many cases where HLA-A24 is the subject in many cases. In Asia, it is expected that the number of cancer patients will further increase due to the extension of life expectancy, and there is a demand for immune cell therapy to increase the number of HLA types to be treated more than ever. The number of HLA-A11 restricted CTL epitopes reported so far is extremely small compared to HLA-A2 and HLA-A24, and most of the reported cases in clinical trials for patients with HLA-A11 Also from the absence, the significance of identification of the HLA-A11-restricted EBV LMP2 and EBV EBNA1 specific CTL epitopes of the present invention seems to be great.

  Among EBV-related malignancies, it is noteworthy that Nasopharyngeal carcinoma (hereinafter referred to as NPC). NPC has a high incidence rate in Southeast Asia including southern China, Hong Kong region, Singapore, Vietnam, Malaysia, Philippines, etc. It also has a relatively high incidence rate in areas such as North Africa. In 2012, 86,691 people were newly diagnosed with NPC all over the world, with 50,828 deaths (GLOBOCAN 2012). In addition, it has been reported that the survival rates at 1, 3, 5, and 10 years after radiation treatment were 89.86%, 60.60%, 47%, and 33.03%, respectively (Non-patent Document 18). Interestingly, the HLA type has been reported as one of the risk of developing NPC (Non-patent Document 17). For example, 50% of A11 carriers, 50% of A2 carriers, and 50% of A24 carriers are the top-ranked HLA-A type in the result of statistically analyzing the frequency of HLA-A type in NPC patients. It is reported that it was 30% and 32% of B40 holders (non-patent document 15). Or, according to the survey results of different research groups, 58% of A11 holders, 53% of A2 holders, 30% of A24 holders, and 43% of B40 holders are reported (Non-Patent Document 16) . Recently, as a result of exhaustive examination of HLA amino acid sequence variants and SNPs using next-generation sequencer technology, HLA-A11 has been reported as an HLA type closely related to the onset of NPC (non-patent literature) 19). As described above, NPC has a high incidence rate of HLA-A11 carriers, and the significance of being able to provide a therapeutic method using the EBV LMP2 and EBV EBNA1 specific CTL epitopes showing the HLA-A11 restriction of the present invention is profound.

  Because NPC is a refractory malignancy, interest in immune cell therapy using EBV-specific CTL activated in vitro as a fourth treatment following surgical procedures, chemotherapy and radiation therapy is highly significant. It has grown and its efficacy has been reported in several clinical trials. However, CTL epitope peptides used in those clinical trials are mainly HLA-A2 and HLA-A24 restricted peptides, and there are few clinical trial reported examples of HLA-A11 restricted CTL epitope peptides. The reason is that the identification of HLA-A11-restricted CTL epitope peptide has hardly been attempted worldwide, and the identification of an EBNA1-derived CTL epitope expressed by NPC is very difficult It can be mentioned.

  In the present invention, using the identification method described later, the identification of a CTL epitope peptide showing restriction to EBV-related malignancies, in particular, HLA-A11, which has the highest prevalence in NPC patients, was investigated.

  EBV has an approximately 170 kbp double-stranded DNA encoding about 85 gene products, but in cancerous areas of NPC, expression of LMP1, LMP2 and EBNA1 which are proteins derived from EBV has been reported. 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 have been identified as cytotoxic T cell epitope peptides. It is an attractive target for However, in previous reports, it has been believed that CTL immune responses are preferentially directed to LMP2 and EBNA1 is not recognized by CTL (Non-patent Documents 22, 23, 24, 25). The reason is that EBNA1 is composed of 641 amino acids in total length, but there is a glycine-alanine repeat (GAr) consisting of about 200 amino acids in its central region (amino acids 101 to 324) It has been found that it interferes with proteasome processing, a major catalytic mechanism that produces MHC class I epitopes (Non-patent Documents 26, 27, 28). Furthermore, it is clear that the same central region interferes with the translation of EBNA1 mRNA, and it has been reported that the expression of EBNA1 protein itself is low (Non-patent Documents 29 and 30).

  On the other hand, CTL epitopes of LMP2 have been reported from multiple research groups. An overlapping peptide library was prepared for LMP2 protein composed of full-length 497 amino acids (derived from B95.8 strain), and CTL epitope search targeting HLA-A2, HLA-A24, HLA-A11, etc. was performed. While many HLA-A2 and HLA-A24 restricted peptides have been identified, only one HLA-A11 restricted peptide has been identified to date (Non-patent Document 15). Although EBV is a latently infected virus in over 90% of adults worldwide, there is a strong regionality in the prevalence of NPC. For example, in areas such as Southeast Asia, North Africa, and Alaska, epidemiological research has revealed that the incidence of NPC is 100 times higher than in other areas (Rickinson, AB, and E. Kieff. 1996. Epstein- Barr virus, p. 2397-2446. In BN Fields, DM Knipe, and PM Howley (ed.), Fields virology, 3rd ed., Vol. 2. Lippincott-Raven, Philadelphia, Pa). The cause of this strong locality has been mentioned factors such as environment, heredity and lifestyle, but differences in the expression level of oncogenic proteins due to differences in EBV strains and differences in immune responses due to amino acid mutations It is also considered that differences in infectious strains may be another important factor involved in the onset of NPC. It should be noted that one of the areas where NPC prevalence is very high is in Guangdong province of China, where one of the widely transmitted EBV strains is GD1 (Zeng MS, Li DJ, Genomic sequence analysis of Epstein-Barr virus strain GD1 from a nasopharyngeal cancer patient. J Virol. 2005 Dec; 79 (24) Liu QL, Song LB, Li MZ, Zhang RH, Wang HM, Ernberg I, Zeng YX. 15323-30). Here, the sequence corresponding to the specific CTL epitope peptide may be different between EBV strains, for example, the B95.8 strain and the GD1 strain, whereby the CTL inducibility and the detection ability with the MHC-tetramer reagent are affected. There is a possibility of receiving. This means that, by using CTLs derived from a mixture of two or more types of peptides for treatment, it is possible to prepare therapeutic CTLs that do not depend on the strain type of the infected person.

On the other hand, it has long been known that CTLs responsible for cell-mediated immunity specifically recognize various viruses and pathogen-infected cells and attack and eliminate them. This means that infected cells present viral or pathogen-derived antigenic peptides via major histocompatibility complex (hereinafter referred to as MHC, in humans, HLA), which T cells recognize, resulting in an immune response. Is caused. Specifically, MHC includes class I and class II, and MHC class I (hereinafter referred to as MHC-I) is expressed on the cell membrane surface of all nucleated cells, and mainly non-self peptides derived from viral proteins as CTL. Present to and activate. On the other hand, 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 non-self peptides are presented to CD4 ⁺ T cells to induce cytokine secretion and antibody production. Do. That is, as a mechanism for protecting a living body from infection, two pathways, MHC-I-CTL and MHC-II-CD4 ⁺ T cells, are provided.

  In 1991, it was reported that peptides derived from MAGE antigen specifically expressed in malignant melanoma were recognized by CTL (Non-patent Document 31). This is an example showing for the first time that MHC-I presents its own antigenic peptide derived from cancer cells to CTL and specifically induces cytotoxic activity. In other words, CTLs can also respond to autoantigens, and the use of their actions has shown the possibility of treating cancer. Since then, identification of proteins specifically expressed in cancer (hereinafter, cancer antigens) and peptide fragments derived from the proteins has been actively promoted. In parallel with these discoveries, development of cancer immunotherapy is actively promoted. Cancer immunotherapy is a method of treating cancer by growing immunocompetent cells such as CTLs that kill and eliminate cancer cells in vitro and in vivo. Several methods can be considered for cancer immunotherapy.

1. 2. Auto-cancer immunotherapy in which a patient's cancer tissue is crushed with liquid nitrogen or the like and the patient is inoculated with the immunostimulant into the patient. 3. Autologous cancerous dendritic cell therapy in which a patient's cancer tissue is crushed with liquid nitrogen etc. and cocultured with dendritic cells isolated and cultured from patient peripheral blood, and then the patient is inoculated with autologous cancer dendritic cell therapy 3. A patient's cancer tissue is crushed with liquid nitrogen or the like, mixed culture with lymphocytes isolated and cultured from the patient peripheral blood, and returned to the patient's body.

  1 to 3 are not necessarily cancer antigen-specific immune responses, and are nonspecific cancer immunotherapies. In addition, there is a disadvantage that surgical treatment such as excision of a cancerous part from a cancer patient is required.

4. 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 patient is inoculated with cancer antigen-specific CTL epitope peptide after pulsing dendritic cells isolated and cultured from patient peripheral blood. A combination therapy of CTL transfer therapy 7.4 and 6 which stimulates and culture lymphocytes isolated and cultured from patient peripheral blood using a cancer antigen-specific CTL epitope peptide to induce specific CTL and return it to the patient's body. The patient is inoculated with a cancer antigen specific CTL epitope peptide and an immunostimulant. A method of inducing specific CTL by stimulating the lymphocytes isolated from the patient's peripheral blood with a cancer antigen-specific CTL epitope peptide, and returning it to the patient's body.
8. Artificial CTL transfer therapy by producing T-cell receptor (TCR) gene from cancer antigen-specific CTL and transferring it to lymphocytes isolated and cultured from patient peripheral blood to produce artificial CTL and return it to the patient's body .

  4-8 all raise and utilize a cancer antigen specific immune response, and are specific cancer immunotherapy. However, in order to carry out these, it is the first requirement to identify a cancer antigen-specific CTL epitope and prove the presence of a CTL that recognizes it.

  As described above, although various cancer immunotherapies that elicit and utilize cancer antigen-specific immune responses have been developed, their therapeutic effects are not necessarily high, and many clinical trials Failing in phase III. Due to these failures, the following cautions can be considered in the implementation of cancer immunotherapy.

(1) Appropriate CTL monitoring Cancer immunotherapy is a treatment that is performed with the expectation of amplification of CTLs that recognize and attack cancer in vivo, and it is important to monitor CTL, which is an effective component. . As a monitoring method of CTL, there are intracellular cytokine staining method, measurement of cytotoxic activity and the like, and these are methods of detecting CTL indirectly. For example, intracellular cytokine staining is a method for measuring the production of IFNγ (interferon gamma) and TNFα (tumor necrosis factor-alpha) in response to peptide stimulation, and may detect responses other than antigenic peptide-specific immune responses. is there. On the other hand, as a more appropriate method, there is a method using an MHC-tetramer reagent. The MHC-tetramer reagent is a reagent in which a ternary complex of MHC and β2-microglobulin (hereinafter, β2m) and a peptide fragment (MHC-monomer) is produced in a test tube and the MHC-monomer is tetramerized.

The reason why this reagent is suitable for monitoring CTL is described below. MHC, which constitutes MHC-monomer, is a diverse molecule, and according to IMGT's HLA database (http://hla.alleles.org/nomenclature/stats.html), HLA-A is 2077 types, HLA-B. There are 2741 types of proteins and 1739 types of proteins for HLA-C (as of October 2014). Moreover, the characteristic of the peptide fragment (8-12 amino acid residue length) to bind | bond also differs by the allyl type of each MHC. That is, there are a huge number of combinations of MHC / peptide complexes, which are only the allyl form of MHC and the types of peptide fragments. On the other hand, CTLs recognize MHC-I presented peptides in TCR. Individual TCRs are produced by rearrangements of TCR genes, and the number of TCR repertoire in one individual is said to be as high as 10 ¹⁸ . That is, the MHC / peptide complex of the target cell and the TCR on the CTL side both have enormous diversity. In addition, individual CTLs generally express one type of TCR on the cell membrane surface, and recognize and activate only specific MHC / peptide complexes thereto. The MHC-tetramer reagent is a reagent utilizing this mechanism. That is, the MHC-tetramer reagent is a reagent that mimics the structure of the MHC / peptide complex on the target cell membrane, whereby only CTLs having a specific TCR can be selectively detected. From this point of view, the MHC-tetramer reagent is considered to be an ideal reagent for monitoring CTL. The reason why the MHC-monomer is tetramerized is to enhance the binding ability with the TCR expressed by CTL and to enable detection with a device such as a flow cytometer.
(2) Mutation of Antigen Presenting Molecules (HLA and β2m) of Cancer Cells Mutations of HLA and β2m of cancer cells are known as a mechanism for immune escape of cancer cells. When these occur, the CTL can not recognize the cancer cell and can not attack because the cancer cell does not present the antigenic peptide. As a mutation of HLA, a LOH mutation (loss of heterozygosity) in which one side of the same locus is deleted is known. Further, mutations of β2m are known as frame shift mutations. With respect to mutations of β2m, attempts have been made to supplement β2m expression by introducing a β2m gene with an adenovirus vector to present an antigen peptide to cancer cells.
(3) Expression of Target Antigen of Cancer Cells There are various types of cancer antigens, and which cancer antigen is expressed varies depending on the type of cancer and individual. For this reason, it is very important to confirm the expression of cancer antigens in patients for cancer immunotherapy. For example, when the cancer antigen derived from the peptide vaccine is not expressed at all in the cancerous part of the patient, the cancer cells can not be targeted, and the therapeutic effect of the cancer peptide vaccine therapy can not be expected. In recent years, in some clinical trials of cancer peptide vaccine therapy, it has been confirmed whether the cancer cells of the patient express the antigen derived from the peptide vaccine, for example, by tissue staining using a cancer antigen specific antibody. . On the other hand, in the case of malignant glioblastoma and the like, it is difficult to confirm the expression of a cancer antigen in a cancer affected area because it is difficult to obtain a cancer cell sample.
(4) Action of immunosuppressive molecule
CTLs are activated when they specifically recognize an antigen peptide presented by target cells, and exert cytotoxic activity. On the other hand, the existence of a molecule that inhibits CTL activation is known, such as TGFβ, PD-1, and CTLA-4. TGFβ is a suppressive cytokine secreted by cancer cells, which inhibits proliferation and differentiation of CTL and CD4 ⁺ T cells. PD-1 and CTLA-4 are both molecules on the cell membrane of T cells, and when bound to a ligand expressed by cancer cells, respectively, a suppressive signal is transmitted to inactivate CTL. In recent years, it has been reported that the action of CTLs is promoted by inhibiting the action of these immunosuppressive molecules by anti-PD-1 antibody and anti-CTLA-4 antibody.
(5) HLA Type of Patient Epitope peptide is basically presented to only one kind of HLA. This is called HLA restriction. Therefore, even if cancer immunotherapy is performed on a patient who does not possess an HLA type capable of presenting an epitope peptide, the cancer cells do not present the epitope peptide in that patient, and thus no therapeutic effect can be expected. That is, the target patients for cancer immunotherapy are limited to the HLA restriction of the epitope peptide used. In fact, in clinical trials for cancer immunotherapy, patients' HLA types are determined in advance by serotyping and genotyping using anti-HLA antibodies, and patients who meet the HLA restriction of the epitope used are tested be registered.

  From the above, in order to obtain a therapeutic effect by cancer immunotherapy, it is necessary to carry out appropriate CTL monitoring, to confirm the expression of an antigen-presenting molecule, to confirm the expression of a cancer antigen in a cancerous area, to immunize. It is important to inhibit the action of the inhibitory molecule and to select a cancer antigen epitope peptide that is compatible with the patient's HLA type. Above all, in order to select a cancer antigen epitope peptide that conforms to the patient's HLA type, it is premised that various HLA restricted epitope peptides have been identified. Therefore, in order to respond to a wide variety of cancer patients, it is desirable that as many HLA-restricted epitope peptides as possible have been identified among multiple cancer antigens. For this reason, identification of the epitope peptide has great significance. HLA-A * 24: 02 is the highest-preserving HLA-A allele in Japanese, and about 60% of them. Therefore, in the present invention, a novel cancer antigen epitope peptide restricted for HLA-A * 24: 02 that can be used for specific cancer immunotherapy, a cancer vaccine and an anticancer agent using the same, and an epitope peptide specific The purpose of the present invention is to provide a reagent for detecting a target CTL.

  CKAP4 is a type II transmembrane protein composed of 63 kDa long and 602 full-length amino acids. From the N-terminal side, there are three regions of the cytoplasmic region consisting of 106 amino acids, the transmembrane region consisting of 21 amino acids, and the extracellular region consisting of 475 amino acids (non-patented) Literature 32). CKAP4 has the function of binding to the endoplasmic reticulum and microtubules in the cytoplasmic domain in the interphase of the cell cycle, and anchoring the endoplasmic reticulum to microtubules (Non-patent Document 33). Before cells enter mitosis, a cysteine residue, which is the 100th amino acid from the N-terminal side of CKAP4, is reversibly palmitoylated (Non-patent Document 34), and this palmitoylation localizes CKAP4. Changes to cell membranes (Non-patent Document 35), and the interaction with microtubules is inhibited. In addition, CKAP4 has been reported to be phosphorylated during mitosis and to lose its ability to bind to microtubules (Non-patent Document 36). On the other hand, it is known that microtubules constitute a spindle having a role of distributing chromosomes at the mitotic stage to properly bisect cells. From these, it is thought that CKAP4 participates in the regulation of normal mitosis by appropriately dissociating from microtubules in the mitotic phase. CKAP4 is also known to act as a receptor for surfactant protein-A (hereinafter referred to as SP-A) responsible for removing surfactant (lung surfactant) on the surface of alveolar cells (non-patented) Literature 32). At the alveolar cell surface, lipid-based surfactants contribute to alveolar spreading by reducing the surface tension of water covering the surface. SP-A has the function of removing a surfactant, and is responsible for maintaining the homeostasis of alveolar cells by functioning together with the receptor CKAP4.

  CKAP4 is expressed even in normal cells, but is highly expressed in various cancer cell lines such as breast cancer, central nervous system tumors, lung cancer, kidney cancer, and malignant melanoma (http://129.187.44.58 : 7070 / NCI60 / protein / show / 10796). In addition, CKAP4 is highly expressed in osteosarcoma patients (http://www.ebi.ac.uk/gxa/experiments/E-MEXP-3628?geneQuery=ENSG00000136026&queryFactorValues=g2_g1&_specific=on), and liver cancer It has been reported that the expression is high in patients (Non-patent Document 37). From these, it is possible that CKAP4 may be an over-expressed cancer antigen that is highly expressed in cancer cells (Non-patent Document 38). For example, Her-2 is known as an overexpression type cancer antigen, and clinical trials of cancer peptide vaccines using Her-2 derived peptides have also been conducted (Non-patent Documents 39 and 40).

  As described above, 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 targeted by the immune system as a cancer antigen, that is, a peptide derived from CKAP4 is presented to HLA on the surface of cancer cells, and CTLs that specifically recognize this attack cancer cells by CTLs. It is unclear as to whether or not it has been done.

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Murray RJ, Kurilla MG, Brooks JM, Thomas WA, Rowe M, Kieff E, Rickinson AB. Identification of the target antigens for T cells response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive J. Exp Med. 1992 Jul 1; 176 (1): 157-68. Steven NM, Leese AM, Annels NE, Lee SP, Rickinson AB. Epitope focusing in the primary cytotoxic T cell response to Epstein-Barr virus and its relationship to T cell memory. J Exp Med. 1996 Nov 1; 184 (5): 1801-13. Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, O'Callaghan CA, Steven N, McMichael AJ, Rickinson AB. Direct visualization of antigen-specific CD8 + T cells during the primary immune response to Epstein-Barr virus In vivo J Exp Med. 1998 May 4; 187 (9): 1395-402. Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen PM, Klein G, Kurilla MG, Masucci MG. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature. 1995 Jun 22; 375 (6533): 685-8. Blake N, Lee S, Redchenko I, Thomas W, Steven N, Leese A, Steigerwald-Mullen P, Kurilla MG, Frappier L, Rickinson A. Human CD8 + T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-) Ala) -containing protein requires exogenous processing. Immunity. 1997 Dec; 7 (6): 791-802. Inhibition of ubiquitin / proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1. Proc Natl Acad Sci US A. 1997 Nov. 11; 94 (23): 12616-21. Yin Y, Manoury B, Fahraeus R. Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1. Science. 2003 Sep 5; 301 (5638): 1371-4. Aplo S, Daskalogianni C, Manoury B, Fahraeus R. Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation revels a close correlation between mRNA translation initiation and antigen presentation. PLoS Pathog. . van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A, Boon T. A gene encoding an antigen recognized by cytolytic T lymphoid cells on a human melanoma. Science. 1991; 254: 1643-1647. Bates SR. P 63 (CKAP 4) as an SP-A receptor: implications for surfactant turnover. Cell Physiol Biochem. 2010; 25: 41-54 Klopfenstein DR, Kappeler F, Hauri HP. A novel direct interaction of endoplasmatic reticulum with microtubules. EMBO J. 1998; 17: 6168-6177. Mundy DI, Warren G. Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein. J Cell Biol. 1992; 116: 135-146. Identification of CKAP4 / p63 as a major substrate of the palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel proteomics Methods. 7: 1378-1388. Phosphorylation controls CLIMP-63-mediated anchoring of the endoplasmic reticulum to microtubules. MoI 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 of cytoskeleton-associated membrane protein 4 and its palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma. Cancer. 2014; 120: 1520-1531. Heemskerk B, Kvistborg P, Schumacher TN. The cancer antigenome. EMBO J. 2013; 32: 194-203. Identification of an immunodominant peptide of HER-2 / neu protooncogene recognized by ovarian tumor-specific cytotoxic lines. J Exp Med. 1995; 181: 2109-2 117. Fisk B, Blevins TL, Wharton JT, Ioannides CG. Results of the first phase 1 clinical trial of the HER-2 / neu peptide (GP2) vaccine in disease-free breast cancer patients: United States Carmichael MG, Benavides LC, Holmes JP, Gates JD, Mittendorf EA, Ponniah S, Peoples GE. States Military Cancer Institute Clinical Trials Group Study I-04. Cancer. 2010; 116: 292-301.

  The present invention has been made in view of such circumstances, and the targets are focused on LMP2 and EBNA1 of EBV, and the purpose is to target LMP2 specific cytotoxic T cell epitope peptide and EBNA1 specific cytotoxicity. A vaccine for treating T cell epitope peptides and treating or preventing EBV-related malignancies and immunodeficiency including NPC using the peptides, a passive immunotherapeutic agent for EBV, and cytotoxic T cells specific for EBV To provide a quantitative method for In addition, the present inventors have intensively studied cancer antigenicity, that is, CTL inducibility, for various CKAP4-derived peptides. As a result, a specific peptide within the extracellular domain of CKAP4, as shown in SEQ ID NO: 43, induces HLA-A * 24: 02-restricted CKAP4 specific CTL, and the specific CTL exhibits cytotoxic activity. Found out. The present invention has been made based on these findings.

The present invention has been made based on such findings, and in a specific aspect, for example, to the following inventions.
[1] An epitope peptide having cytotoxic T cell-inducing activity specific to Epstein-Barr virus (EBV), wherein the epitope peptide comprises SEQ ID NO: 1, SEQ ID NO: 2, sequence An epitope peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28;
[2] An epitope peptide having cytotoxic T cell-inducing activity specific to Epstein-Barr virus (EBV), wherein the epitope peptide comprises SEQ ID NO: 1, SEQ ID NO: 2, sequence No. 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 27 or in the amino acid sequence described in SEQ ID NO: 28, one or more amino acids are substituted or deleted , Inserted and / or added, epitope peptide,
[3] A nucleic acid encoding the epitope peptide according to any one of [1] to [2],
[4] An expression vector comprising a nucleic acid encoding the epitope peptide according to any one of [1] to [2],
[5] A vaccine for treating or preventing EBV infection or EBV-positive cancer, comprising the epitope peptide according to any one of [1] to [2] as an active ingredient,
[6] The vaccine according to [5], wherein the EBV is selected from an AKata strain, a GD1 strain, a GD2 strain, a HKNPC1 strain, an AG876 strain, a Mutu strain and a B95.
[7] A vaccine for treating or preventing EBV infection or EBV-positive cancer, comprising the nucleic acid or expression vector according to any one of [7] [3] to [4] as an active ingredient,
[8] The vaccine according to [7], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.
[9] [1] to [2] a vaccine for treating or preventing EBV infection or EBV virus-positive cancer, comprising as an active ingredient an antigen-presenting cell having HLA-presented antigen-presenting cells. ,
[10] The vaccine according to [9], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.
[11] [1] to [2] or the EBV-specific cytotoxic T cell obtained by stimulating peripheral blood lymphocytes with antigen-presenting cells presenting the epitope peptide to HLA. Passive immunotherapeutic agents for EBV, including
Contacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the epitope peptide according to any one of [12] [1] and [2] with peripheral blood lymphocytes, A conjugate in which a cytotoxic T cell is bound to the major histocompatibility complex and / or a major histocompatibility complex-tetramer is formed, and a cytotoxic T cell obtained by isolation from the conjugate is obtained Passive immunotherapeutic agents for EBV, including
[13] A step of stimulating peripheral blood from a subject with the epitope peptide of any one of [1] to [2],
Obtaining EBV-specific cytotoxic T cells produced by the above steps, and measuring cytokines and / or chemokines and / or cell surface molecules produced by the obtained cytotoxic T cells, Method for quantifying specific cytotoxic T cells,
[14] [1]-[2] mixing the epitope peptide described in any one of [1] to [2] with the major histocompatibility complex and the β2-microglobulin, and preparing the major histocompatibility complex-tetramer and the subject A method of quantifying cytotoxic T cells specific for EBV in the peripheral blood, comprising the step of contacting the peripheral blood with the
[15] [1]-[2], a method for inducing cytotoxic T cells specific to EBV, which comprises the step of bringing an antigen-presenting cell into contact with the epitope peptide described in any one of [15] [1]-[2].
[16] A kit for inducing cytotoxic T cells, which comprises the epitope peptide of any one of [1] to [2] as a component.
[17] A method for producing an EBV-specific CTL, comprising the step of contacting a peripheral blood mononuclear cell with the peptide according to any one of [1] to [2] in a medium containing plasma,
[18] A CTL epitope peptide specific for CKAP4, which comprises the amino acid sequence set forth in SEQ ID NO: 43,
[19] A CTL epitope peptide specific for CKAP4, wherein one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 43,
[20] HLA-A * 24: 02 molecule-restricted antigen peptide which specifically recognizes a cell presenting a complex with HLA-A * 24: 02 molecule on the cell surface The epitope peptide according to any one of [18] or [19], which is characterized by inducing CTL having
[21] A nucleic acid encoding the epitope peptide according to any of [18] to [19], an expression vector comprising a nucleic acid encoding the epitope peptide according to any of [22] [18] to [19],
[23] A vaccine for treating and preventing cancer, which comprises the epitope peptide according to any one of [18] to [19] as an active ingredient,
[24] A vaccine for treating or preventing cancer, which comprises the nucleic acid according to any one of [21] to [22] as an active ingredient,
[25] A vaccine for treating or preventing cancer, which comprises, as an active ingredient, an antigen-presenting cell in which the epitope peptide according to any one of [18] to [19] is presented to HLA.
[26] [18] to [19] or the CKAP4-specific CTL obtained by stimulating peripheral blood lymphocytes with antigen-presenting cells presenting the epitope peptide to HLA as an active ingredient Passive immunotherapeutic agents for cancer treatment, including
[27] A major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the epitope peptide of any one of [18] to [19] and a peripheral blood lymphocyte are brought into contact with each other, A cancer treatment comprising, as an active ingredient, CTL obtained by forming a conjugate in which CTL binds to the major histocompatibility complex and / or major histocompatibility complex-tetramer, as an active ingredient Passive immunotherapeutic agent, for
[28] Stimulating peripheral blood from a subject with the epitope peptide of any one of [18] to [19],
Obtaining a CTL specific for CKAP4 generated by the above steps, and measuring a cytokine and / or a chemokine and / or a cell surface molecule produced by the obtained CTL,
A method of quantifying CTL specific for CKAP4, including
[29] A process for preparing a major histocompatibility complex-tetramer from the epitope peptide according to any of [18] to [19], and a major histocompatibility complex-tetramer and peripheral blood from a subject A method of quantifying CTL specific for CKAP4 in the peripheral blood, comprising the step of
[30] A method for inducing CKAP4 specific CTL, comprising the step of contacting the epitope peptide according to any one of [18] to [19] with peripheral blood mononuclear cells from a subject,
[31] Stimulating peripheral blood lymphocytes with the epitope peptide according to any of [18] to [19] or an antigen-presenting cell presenting the epitope peptide to HLA, and obtaining CKAP4 specific CTL , A method of producing a passive immunotherapeutic agent for cancer treatment,
Contacting the major histocompatibility complex and / or the major histocompatibility complex-tetramer prepared from the epitope peptide according to any of [32] [18] to [19] with peripheral blood lymphocytes ,
Forming a conjugate in which CTL binds to the major histocompatibility complex and / or major histocompatibility complex-tetramer, and obtaining a CTL obtained by isolation from the conjugate. Method of producing a passive immunotherapeutic agent for cancer treatment,
[33] An antibody specific to a major histocompatibility complex prepared from the epitope peptide according to 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] The 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] The use according to [A3], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain,
[A5] Use of an antigen-presenting cell which presented the epitope peptide according to any one of [1] to [2] to HLA in the manufacture of a medicament for treating or preventing EBV infection or EBV virus-positive cancer ,
[A6] The use according to [A5], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain,
[A7] Stimulation of peripheral blood lymphocytes with the epitope peptide according to any one of [1] or [2] or an antigen-presenting cell presenting the epitope peptide to HLA for the production of a passive immunotherapeutic agent for EBV Use of EBV-specific cytotoxic T cells obtained
[A8] Major histocompatibility complex and / or major histocompatibility complex-tetramer prepared from the epitope peptide according to any one of [1] to [2] in the manufacture of a passive immunotherapeutic agent for EBV Is brought into contact with the peripheral blood lymphocytes to form a conjugate in which a cytotoxic T cell is bound to the major histocompatibility complex and / or the major histocompatibility complex-tetramer, and a single conjugate is isolated from the complex. Use of 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 presenting to HLA the epitope peptide according to any one of [18] to [19] 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 treated with the epitope peptide of any of [18]-[19] or an antigen-presenting cell presenting the epitope peptide to HLA. Use of CKAP4 specific CTL obtained by stimulation
[A13] A major histocompatibility complex and / or a major histocompatibility antigen prepared from the epitope peptide according to any one of [18] to [19] in the manufacture of a passive immunotherapeutic agent for cancer treatment The complex-tetramer is brought into contact with peripheral blood lymphocytes to form a conjugate in which a CTL is bound to the major histocompatibility complex and / or the major histocompatibility complex-tetramer, and a single complex is isolated from the conjugate. Use of CTL obtained separately,
[B1] A vaccine composition comprising, as an active ingredient, the epitope peptide of any one of [1] to [2], for treating or preventing EBV infection or EBV-positive cancer.
[B2] 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, as an active ingredient, the nucleic acid or expression vector according to any one of [3] to [4], for treating or preventing EBV infection or EBV-positive cancer.
[B4] The vaccine composition according to [B3], wherein EBV is selected from an AKata strain, a GD1 strain, a GD2 strain, a HKNPC1 strain, an AG876 strain, a Mutu strain and a B95.8 strain,
[B5] For containing, as an active ingredient, an antigen-presenting cell on which the epitope peptide described in any one of [1] to [2] is presented to HLA for treating or preventing EBV infection or EBV virus-positive cancer Vaccine composition,
[B6] The vaccine composition according to [B5], wherein the EBV is selected from the strain AKata, the strain GD1, the strain GD2, the strain HKNPC1, the strain AG876, the strain Mutu and the strain B95.8;
[B7] Obtained by stimulating peripheral blood lymphocytes with the epitope peptide according to any of [1] or [2] or an antigen-presenting cell presenting the epitope peptide to HLA for passive immunotherapy against EBV A composition comprising EBV-specific cytotoxic T cells,
[B8] Major histocompatibility complex and / or major histocompatibility complex-tetramer prepared from the epitope peptide according to any one of [1] to [2] for passive immunotherapy against EBV Contact with peripheral blood lymphocytes to form a conjugate in which cytotoxic T cells are bound to the major histocompatibility complex and / or the major histocompatibility complex-tetramer, and isolated from the conjugate A composition comprising cytotoxic T cells obtained by
[B9] A vaccine composition comprising, as an active ingredient, the epitope peptide of any one of [18] to [19], for treating or preventing cancer.
[B10] A vaccine composition comprising, as an active ingredient, the nucleic acid or expression vector according to any one of [21] to [22] for treating or preventing cancer.
[B11] A vaccine composition comprising, as an active ingredient, an antigen-presenting cell in which the epitope peptide according to any one of [18] to [19] is presented to HLA for treatment or prevention of cancer.
[B12] for stimulating peripheral blood lymphocytes with the epitope peptide of any one of [18]-[19] or antigen-presenting cells presenting the epitope peptide to HLA for treating cancer by passive immunotherapy A composition comprising, as an active ingredient, the CKAP4 specific CTL obtained by
[B13] A major histocompatibility complex and / or a major histocompatibility complex prepared from the epitope peptide according to any one of [18] to [19] for treating cancer by passive immunotherapy -Contacting tetramer with peripheral blood lymphocytes to form a conjugate in which CTL binds to the major histocompatibility complex and / or the major histocompatibility complex-tetramer, and isolated from the conjugate A composition comprising the obtained CTL as an active ingredient,
A method for treating or preventing EBV infection or EBV-positive cancer, comprising the step of administering the epitope peptide of any one of [C1] [1] and [2] to an individual in need thereof ,
[C2] The method according to [C1], wherein EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain,
For treating or preventing EBV infection or EBV-positive cancer, comprising the step of administering the nucleic acid or expression vector according to any one of [C3] [3] to [4] to an individual in need thereof [C4] The method according to [C3], wherein [C4] EBV is selected from AKata strain, GD1 strain, GD2 strain, HKNPC1 strain, AG876 strain, Mutu strain and B95.8 strain,
An EBV-infected or EBV-positive cancer comprising the step of administering to an individual in need thereof an antigen-presenting cell presenting the epitope peptide according to any one of [C5] [1] and [2] to HLA. Methods 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,
EBV-specific cytotoxic T cells obtained by stimulating peripheral blood lymphocytes with the epitope peptide according to any of [C7] [1] and [2] or an antigen-presenting cell presenting the epitope peptide to HLA. Passive immunotherapy for EBV, comprising the step of administering to an individual in need thereof,
Contacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the epitope peptide according to any of [C8] [1] and [2] with peripheral blood lymphocytes ,
Forming a conjugate in which a cytotoxic T cell is bound to the major histocompatibility complex and / or the major histocompatibility complex-tetramer;
Passive immunotherapy for EBV, comprising the step of administering cytotoxic T cells obtained by isolation from said conjugate to an individual in need thereof,
A method for treating or preventing cancer, comprising the step of administering the epitope peptide of any one of [C9] [18] to [19] to an individual in need thereof;
A method for cancer treatment or prevention, comprising the step of administering the nucleic acid or expression vector according to any one of [C10] [21] to [22] to an individual in need thereof.
A method for treating or preventing cancer, which comprises the step of administering to a subject in need thereof an antigen-presenting cell presenting the epitope peptide according to any of [C11] [18] to [19] to an HLA. ,
The epitope peptide described in any one of [C12] [18] to [19] or a CKAP4 specific CTL obtained by stimulating peripheral blood lymphocytes with antigen-presenting cells presenting the epitope peptide to HLA, Passive immunotherapy for cancer treatment, including the step of administering to an individual in need
Contacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the epitope peptide according to any one of [C13] [18] to [19] with peripheral blood lymphocytes ,
Forming a conjugate in which CTL binds to the major histocompatibility complex and / or the major histocompatibility complex-tetramer;
Passive immunotherapy for cancer treatment, comprising the step of administering CTLs isolated and obtained from the conjugate to an individual in need thereof.

FIG. 6 shows a representative gel filtration column analysis when MHC-monomer formation is observed. It is a figure which shows the folding test result of a EBV LMP2 specific CTL epitope candidate peptide. It is a figure which shows the folding test result of EBV EBNA1 specific CTL epitope candidate peptide. It is a figure which shows the examination result of the intracellular IFNgamma production cell quantification method using a control peptide. It is the figure which confirmed EBV LMP2 specific CTL induction by the intracellular IFNgamma production cell quantification method, and quantified the ratio of the IFNgamma production live cell which exists in a CD8 positive cell. LMP2-specific CTL induction was confirmed by the intracellular IFNγ-producing cell assay (donor ID * 11-11). Dot plot developed plot of fluorescence intensity for IFNγ on CD8 on X axis and IFNγ on Y axis, showing the result of quantification of intracellular IFNγ producing cells by restimulation with the same peptide used for induction FIG. It is a figure which shows the detection result of LMP2 specific CTL by the produced | generated MHC-tetramer reagent (donor ID * 11-11). It is a figure which shows the detection result (1) of EBNA1 specific CTL by the produced MHC-tetramer reagent. It is a figure which shows the detection result (2) of EBNA1 specific CTL by the produced MHC-tetramer reagent. It is a figure which shows the confirmation result of EBNA1 specific CTL induction by intracellular IFNgamma production cell quantification method (donor ID * 11-8). It is a figure which shows the change of MHC affinity by deleting the amino acid of the N terminal and C terminal of ASS (10 mer). It is a figure which shows the change of CTL inducibility by deleting the amino acid of the N terminal and C terminal of ASS (10 mer) (donor ID * 11-11). It is a figure which shows the change of TCR binding property by deleting the amino acid of the N terminal and C terminal of ASS (10 mer) (donor ID * 11-11). FIG. 2 shows amino acid mutation comparison between ASS (10 mer) and HRG EBV strains. It is a figure which shows the result of a sequence of ASS (10 mer) CTL epitope. It is a figure which shows the inducibility difference by the amino acid mutation of ASS (10 mer) (donor ID: * 11-11). It is a figure which shows the change of TCR binding property by the amino acid substitution of ASS (10 mer) (donor ID: * 11-11). It is a figure which shows the result of mass culture (1) of CTL using a culture | cultivation bag. It is a figure which shows the result of mass culture (2) of CTL using a culture | cultivation bag. It is a figure which shows the result of mass culture (3) of CTL using a culture | cultivation bag. It is a figure which shows the example of a representative gel filtration column analysis when MHC-monomer formation is recognized. It is a figure which shows the folding test result of a CKAP4 specific CTL epitope candidate peptide. It is a figure which shows the induction | guidance result of CKAP4 specific CTL (sample number; A24-37, 1st step). It is a figure which shows the induction | guidance result of CKAP4 specific CTL (sample number; A24-37, 2nd step, lane 7). It is a figure which shows the induction result of CKAP4 specific CTL (sample number; A24-39, 1st step). It is a figure which shows the induction | guidance result of CKAP4 specific CTL (sample number; A24-39, 2nd step, lane 10-11). It is a figure which shows the quantification result of IFNgamma production cell in CKAP4 specific CTL.

  The present invention will be described in more detail.

[Epitope peptide]
The term "peptide" as used in the present invention means a molecular chain of linear amino acids which have physiological activity and are mutually linked by a peptide bond between the α-amino group and carboxyl group of adjacent amino acid residues. Peptides do not imply specific lengths and can be of various lengths. In addition, it may be uncharged or in the form of a salt, and in some cases, it may be modified by glycosylation, amidation, phosphorylation, carboxylation, phosphorylation and the like. Furthermore, the epitope peptide of the present invention does not substantially alter the physiological activity and the immune activity, and one or several (for example, 1 to 10), unless having an adverse activity when administered Also included in the present invention are peptides in which insertions, additions, substitutions, deletions and the like of amino acids or amino acid analogs occur. The purpose of changing such amino acids is, for example,
1. Changes to enhance affinity with HLA (Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topianian SL, Restifo NP, Dudley ME, Schwarz SL, Spiess PJ, Wunderlich JR, Parkhurst MR, Kawakami Y, Immunological and therapeutic evaluation of a synthetic peptide vaccine for the treatment with patients with metastatic melanoma. Nat Med. 1998; 4: 321-327, Berzofsky JA, Ahlers JD, Belyakov IM. Strategies and optimizing new generation vaccines. Nat Rev Immunol. 2001; 1: 209-219),
2. Alteration to improve the recognition of TCR (Fong L, Hou Y, Rivas A, Benike C, Yuen A, Fisher GA, Davis MM, Engleman EG. Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc. Natl Acad Sci US A. 2001; 98: 8809-8814, Rivoltini L, Squarcina P, Loftus DJ, Castelli C, Tarsini P, Mazzocchi A, Rini F, Viggiano V, Belli F, Parmiani G. A superagonist variant of peptide MART1 Cancer Res. 1999; 59: 301-306), / Melan A27-35 elicits anti-melanoma CD8 + T cells with enhanced functional characteristics: implications for more effective immunotherapy.
3. Changes to avoid metabolism due to peptide degradation enzymes in serum (Berzofsky JA, Ahlers JD, Belyakov IM. Strategies for designing and optimizing new generation vaccines. Nat Rev Immunol. 2001; 1: 209-219, Parmiani G, Castelli J Natl Cancer Inst. 2002; 94: 805-818, Brinkerhoff LH C, Dalerba P, Mortarini R, Rivoltini L, Marincola FM, Anichini A. Cancer immunotherapy with peptide-based vaccination: What have we achieved? Terminal modifications inhidative proteolytic degradation of an immunogenic MART-1 (27-35) peptide: implications for peptone Int vices. Kalashnikov VV, Thompson LW, Yamshchikov GV, Pierce RA, Galavotti HS, Engelhard VH, Slingluff CL Jr. 1999; 83: 326-334) and the like.

  Also included are those in which an additional amino acid sequence intervenes at the N-terminus or C-terminus of the peptide for such purposes. In addition, the peptide of the present invention can be used in the form of a saccharide, polyethylene glycol, a complex to which a lipid or the like is added, a derivative by a radioactive isotope or the like, or a polymer. Examples of the amino acid analogues include N-acylated products, O-acylated products, esterified products, acid amidized products, alkylated products of various amino acids and the like.

  In addition, as long as CTL can specifically recognize a cell that presents a ternary complex of an HLA molecule and β2-microglobulin and an epitope peptide on the cell surface, the N-terminus or free amino group of the antigen peptide Formyl group, acetyl group, t-butoxycarbonyl (t-Boc) group, etc. may be bonded to the C-terminal or free carboxyl group of the antigen peptide, such as methyl group, ethyl group or t-butyl group. A group, a benzyl group or the like may be bonded.

  Furthermore, the epitope peptide of the present invention may be one that has been subjected to various modifications that can facilitate its introduction into the living body. PT (Protein Transduction) domain is famous as an example of various modifications that can facilitate introduction into a living body. The PT domain of HIV is a peptide composed of amino acids 49 to 57 of Tat protein (Arg Lys Lys Arg Arg Gln Arg Arg Arg). 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, Enhanced uptake of a heterologous protein with an HIV-1 Tat protein transduction domains (PTD) at both termini. Mol cells. 2003; 16: 385-391, Kim DT, Mitchell DJ, Brockstedt DG, Fong L, Nolan GP Introduction to soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide. J Immunol. 1997; 159: 1666-1668), Fathman CG, Engleman EG, Rothbard JB.

  Most antigens presented via HLA class I molecules are degraded by proteasome in the cytoplasm, transferred to TAP (transporter in antigen processing), and HLA-associated to TAP in rough endoplasmic reticulum. It binds to a complex of class I molecules and β2-microglobulin and is transported to the cell surface by exocytosis via the Golgi apparatus. By presenting a chaperone HSP (heat shock prtein) 70 or HSP 90 that acts on the series of antigen presentation pathways, or gp96 with a target peptide or protein, it is possible to efficiently present an antigen ( Basu S, Binder RJ, Ramalingam T, Srivastava PK. CD 91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity. 2001; 14: 303-313).

[Nucleic acid encoding epitope peptide]
The nucleic acid encoding the epitope peptide is important for producing the epitope peptide in a host using genetic recombination techniques. In this case, since codon usage of amino acid codons differs between hosts, it is desirable to change the codons of amino acids so as to be compatible with the codon usage of the host to be produced. The nucleic acid encoding the epitope peptide is also important as a vaccine, and can be transferred as a naked nucleic acid or can be transferred using an appropriate viral or bacterial vector (Berzofsky JA, Ahlers JD, Janik J, Morris J, Oh C, 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). A suitable bacterial vector is a bacterium of the genus Salmonella spp. Suitable viral vectors are, for example, retroviral vectors, EBV vectors, vaccinia vectors, Sendai virus vectors, lentiviral vectors. One example of a suitable vaccinia vector is a modified vaccinia ankara vector.

[Selection of CTL Epitope Candidate Peptides]
1. Analysis using a computer prediction algorithm The EBV-specific CTL epitope candidate peptides of the present invention are different from the amino acid sequences of LMP2 and EBNA1 from 8 to 10 amino acids having a binding motif for the target HLA class I molecule. Of multiple epitopes available on the Internet (Pingping Guan, Irini A. Doytchinova, Christianna Zygouri, and Darren R. Flower MHCPred: a server for quantitative prediction of peptide?) MHC binding Nucleic Acids 31.3621-3624, Karosiene E, Lundegaard C, Lund O, Nielsen M. NetMHCcons: a consensus method for the major histocompatibility complex class I predictions. Immunologys. 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. epicytosis discovery. Immunology. 2014 Jan; 141 (1): 18-26. Can be selected by collating.

2. Examination using anchor motif
The HLA class I molecules are mainly HLA-A, HLA-B, and HLA-C, and the epitope peptide presented upon binding to these consists of 8 to 10 amino acids. The 2nd and the 9th or 10th amino acids from the N-terminal side of the epitope peptide are the most important amino acids for binding to HLA class I molecules, and are called anchor motifs. It is reported that this anchor motif is different depending on the type of each HLA class I molecule. For example, as a peptide that binds to HLA-A2 most studied in the world, a peptide in which Leu is arranged at the second position from the N-terminus and Leu or Val is arranged at the 9th or 10th position And peptides consisting of 9 to 10 amino acids are most well known (T Sudo, N Kamikawaji, A Kimura, Y Date, CJ Savoie, H Nakashima, E Furuichi, S Kuhara, and T Sasazuki Differences in MHC class I self peptide repertoires among HLA-A2 subtypes J. Immunol., 1995; 155: 4749-4756). In addition, HLA-A11 which is frequently found in Southeast Asian races including Japanese (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, Bertolletti A, Grotenbreg GM. Conditional ligand 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. As the peptide binding to the peptide, any of Ile, Met, Ser, Thr, or Val is placed at the second position from the N-terminal, and either Lys or Arg is placed at the 9th or 10th position The peptide consisting of 9 to 10 amino acids is most well known (Rapin N, Hoof I, Lund O, Nielsen M. The MHC motif viewer: a visualization tool for MHC binding motifs. Curr Protoc Immunol. 2010 Feb; Chapter 18: Unit 18.17.). From the amino acid sequence of the protein, 8 to 10 amino acid sequences having this anchor motif can be searched to select CTL epitope candidate peptides.

3. Preparation of peptide library
Among the proteins constituting EBV, a peptide library consisting of about 20 amino acid sequences covering the entire target protein is synthesized. Of about 20 amino acids, about 10 amino acid sequences make a library so as to overlap with the peptides before and after. This makes it possible to exhaustively search the entire protein, and once a library is prepared, it becomes possible to comprehensively examine HLA restriction.

[Synthesis of peptide]
The CTL epitope candidate peptides shown in SEQ ID NOs: 1 to 35 of the present invention can be prepared by various conventional peptide synthesis methods. For example, 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. In addition, synthesis by a commercially available chemical synthesizer (for example, peptide synthesizer from Applied Biosystems) is also possible.

[Study of CTL epitope candidate peptides]
The CTL epitope candidate peptide selected by the above-mentioned method may not necessarily be a CTL epitope peptide, and may be an EBV LMP2 specific CTL epitope peptide and an EBV EBNA1 specific CTL epitope peptide for the first time after the following examination.

(1) Examination using cultured cell lines The peptide fragments generated by proteolysis by proteasome are introduced into the endoplasmic reticulum by TAP (transporter associated with antigen processing) molecules, and HLA class I molecules and β2-microglobulin It binds to the complex and is transported to the cell membrane surface. The TAP gene deficient cell line deficient in this TAP molecule can not express a peptide fragment, which is a degradation product of an endogenous protein, on the cell membrane surface. In addition, the human lymphoblastoid cell line T2, which is a typical TAP gene-deficient cell line, or an HLA molecule of a cell line (T2-A11) in which an HLA-A11 molecule has been introduced into T2 has expression on the cell membrane surface. Very unstable. However, when bound to an externally supplied peptide, HLA molecules stabilize on the cell membrane surface. Using this property, the TAP gene-deficient cell line can be used in experiments to verify the binding between HLA molecules and externally supplied peptides. Specifically, after a mixed culture of a TAP gene-deficient cell line and a CTL epitope candidate peptide, the cells are stained with an anti-HLA antibody, and the change in the expression intensity of the HLA molecule is calculated by flow cytometry. The binding properties of CTL epitope candidate peptides can be examined. When a CTL epitope candidate peptide is bound to an HLA molecule expressed by a TAP gene-deficient cell line, the complex of the HLA molecule and the peptide is stabilized on the cell membrane surface, and when stained with an anti-HLA antibody, expression of the HLA molecule is enhanced Is observed. On the other hand, when the added CTL epitope candidate peptide does not show binding to the HLA molecule, the HLA molecule on the cell membrane surface is unstable, and even if it is stained with the anti-HLA antibody, the 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.

(2) Folding test
The MHC-tetramer reagent is a reagent in which a three-way complex (MHC-monomer) of MHC (HLA in case of human) and β2-microglobulin and a peptide fragment is produced in a test tube, and the reagent is obtained by tetramerizing the MHC-monomer. is there. The MHC-tetramer reagent is the only reagent that can selectively detect antigen-specific cytotoxic T cells (CTL) that show MHC restriction. In addition, MHC-tetramer reagent can not only be able to quantify the number of CTL by flow cytometry analysis after co-staining with anti-CD (cluster of differentiation) antibody or anti-cytokine antibody etc., but also its activation state and differentiation stage. It is possible to evaluate every single cell. The first step in the preparation of the MHC-tetramer reagent begins by folding the raw materials MHC, β2-microglobulin and peptide in a suitable solution in a test tube. In the folding solution, a three-way complex (MHC-monomer) is formed by the association reaction of the three raw materials. At this time, if the binding strength between the MHC and the peptide is high, this association reaction proceeds smoothly, and analysis with a gel filtration column makes it possible to detect a complex of three types of raw materials (MHC-monomer). On the other hand, when there is no binding ability between MHC and peptide, MHC-monomer is hardly detected. Therefore, it is possible to verify the binding property and stability of the MHC and the peptide by analyzing the folding solution with time or by performing heat treatment or the like.

(3) Detection of epitope peptide-specific CTL (3-1) Method of determining epitope peptide
Peripheral blood mononuclear cells (PBMC) isolated from a person with a history of EBV infection or T cells isolated from PBMC are suspended in an appropriate medium at a cell concentration of 0.1 to 2 × 10 ⁶ / mL. To this is added 1 × 10 ⁵ / mL of EBV-infected cells which have been previously separated and cultured from the same person, and cultured at 37 ° C. for 7 days in a 5% carbon dioxide (CO ₂ ) thermostat. After 7 days of culture, EBV-infected cells and interleukin 2 (IL-2) are added, and thereafter, CTL is induced by repeating stimulation with EBV-infected cells and IL-2 weekly. Whether or not the thus derived CTL has specificity for the epitope candidate peptide can be determined by the MHC-tetramer method, ELISPOT 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. Shevach, Warren Strober 6.19 ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells 6.24 Detection of Intracellular Cytokines by Flow Cytometry published by John Wileyley & Sons, Inc.).

(3-2) Epitope peptide determination method 2
PBMCs isolated from persons with EBV infection history are suspended in an appropriate medium at a cell concentration of 0.1 to 2 × 10 ⁶ / mL, to which any one of the epitope candidate peptides is at a concentration of 0.01 to 100 μg / mL Add. Incubate at 37 ° C. in a 5% CO ₂ thermostat and after 2 days add IL-2. Thereafter, CTLs are induced by repeating stimulation with the peptide and IL-2 once a week or once every two weeks. Whether or not the thus derived CTL is specific for the epitope candidate peptide is determined by the MHC-tetramer method, ELISPOT assay, chromium release assay, intracellular cytokine staining method or the like.

(3-3) Epitope peptide determination method 3
PBMCs isolated from persons with EBV infection history were suspended in an appropriate medium at a cell concentration of 0.1 to 2 × 10 ⁶ / mL, and the peptide library synthesized thereon was pooled to an appropriate number (for example, 10 each) Add things. Incubate at 37 ° C. in a 5% CO ₂ thermostat and after 2 days add IL-2. Thereafter, CTLs are induced by repeating stimulation with the pool peptide and IL-2 once a week or once every two weeks. Examination of whether or not the thus derived CTL is specific for the epitope candidate peptide is determined by ELISPOT assay, chromium release assay, intracellular cytokine staining method or the like. It is possible to select a peptide having CTL inducibility by adding one peptide at a time to the pooled peptides showing good results and repeating the above experiment. The reacted peptides are sequentially shortened, and finally an epitope peptide consisting of 8 to 10 amino acids is obtained to obtain the epitope peptide of the present invention.

Preparation of EBV LMP2 and EBNA1 Specific MHC-Monomer and MHC-Tetramer Reagents
MHC-monomer and MHC-tetramer reagents using EBV LMP2-specific CTL epitope candidate peptides and EBV EBNA1-specific CTL epitope candidate peptides can be prepared according to known methods (US Patent Number 5,635, 363, French Application Number FR9911133) . MHC-, a complex of three of HLA class I molecule, β2-microglobulin and LMP2-specific CTL epitope candidate peptide of the present invention or EBNA1-specific CTL epitope candidate peptide purified from recombinant host for protein expression The monomers are formed in the folding solution. A biotin binding site is previously added to the C terminus of the recombinant HLA class I molecule, and biotin is added to this site after MHC-monomer formation. The MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4. The differentiation stage of CTL can be examined by using an MHC-tetramer reagent and an antibody against cell surface proteins (CD62L, CCR7, CD45RA, etc.) (Seder RA, Ahmed R. Similarities and differences in CD4 + and CD8 + effector and memory T cell generation. Nat Immunol. 2003; 4: 835-842.). Alternatively, it can be used for functional evaluation of CTL in combination with intracellular cytokine staining. For example, in hepatitis C, CTL against HCV (Hepatitis C virus) exists as one of the causes of sustained infection, but CTL does not produce cytokines or the like, or the proportion of CTL produced is extremely low. It has been reported that it may be chemically unresponsive (anergy) (Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, Walker B, Sullivan J, Phillips R, Pape GR Klerman P. Sustained dysfunction of antiviral CD8 + T lymphocytes after infection with hepatitis C virus. J Virol. 2001; 75: 5550-5558. In addition, for CMV-specific CTL after bone marrow transplantation, it is thought that not only examining the presence or absence of the CTL but also examining the strength of cytokine production ability is effective for measuring the timing of antiviral drug administration etc. (Ozdemir E, St John LS, Gillespie G, Rowland-Jones S, Champlin RE, Molldrem JJ, Komanduri KV. Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8. 2002; 100: 3690-3697.). By identifying such a specific CTL epitope peptide and manufacturing an MHC-tetramer reagent, it becomes possible to quantify and characterize specific CTL, and a large contribution can be made in obtaining diagnostic information.

[Active immune vaccine]
Peptide Vaccines The CTL epitope peptides of the invention can be used as peptide vaccines 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 are grown in the body to prevent infection and to treat infection and treatment for EBV positive tumors. It can be useful. The epitope peptide to be used may be used alone or in combination of two or more peptides depending on the purpose of 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. The antigen-presenting cell on which the CTL epitope peptide is presented is
1. CTL epitope peptide-pulsed antigen-presenting cells in which antigen-presenting cells and CTL epitope peptide are mixed for 30 minutes to 1 hour in a suitable culture solution. 2. Cells in which an antigen-presenting cell is presented with a CTL epitope peptide by gene transfer or the like using a nucleic acid encoding the CTL epitope peptide. It means an artificial antigen presenting cell having an antigen presenting ability prepared artificially. The antigen-presenting cells mean, for example, dendritic cells, B cells, macrophages, certain T cells, etc., but are cells expressing on their cell surface an HLA molecule to which the peptide can bind, such as CTL. It means one having stimulation ability. An artificially prepared antigen-displaying cell having an antigen-displaying ability is prepared, for example, by immobilizing a ternary complex of an HLA molecule, a CTL epitope peptide and β2-microglobulin on beads such as a lipid bilayer membrane, plastic or latex. Or a costimulatory molecule such as CD80, CD83 or CD86 that can stimulate CTL, or fix an antibody that acts on an agonistic effect on CD28, which is a T cell side ligand that binds to the costimulatory molecule. (Oelke M, Maus MV, Didiano D, June CH, Mackensen A, Schneck JP. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med. 2003; 9: 619-624, Walter S, Herrgen L, Schoor O, Jung G, Wernet D, Buhring HJ, Rammensee HG, Stevanovic S. 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 efficient histocompatibility minor-histocompatibility antigen-specific cytotoxic T lymphocytos. -226).

Genetic Vaccine The nucleic acid of the CTL epitope peptide of the present invention can be used as a DNA vaccine or a recombinant viral vector vaccine in active immunotherapy. In this case, it is desirable to change the nucleic acid sequence of the CTL epitope peptide to a recombinant vaccine or codon usage compatible with the host producing the recombinant virus vaccine (Casimiro, DR et al. Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid , Recombinant-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene J. Virol., 2003; 77: 6305-6313, 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).

  Vaccines comprising the CTL epitope peptides of the present invention, or antigen presenting cells on which CTL epitope peptides are presented can be prepared using methods known in the art. For example, such 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 the form of a neutral or salt, for example, pharmaceutically acceptable salts include inorganic salts such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid and tartaric acid . In addition, antigen-presenting cells on which the CTL epitope peptide of the present invention is presented are pharmaceutically acceptable and an excipient compatible with the peptide or the activity of the cells, such as water, saline, dextrose, ethanol, It can be used by mixing with glycerol, DMSO (dimethyl sulfoxide), and other adjuvants or the like, or a combination thereof. Furthermore, if necessary, adjuvants such as albumin, a wetting agent and an emulsifying agent may be added.

  The vaccine of the present invention can be administered by parenteral administration and oral administration, but parenteral administration is generally preferred. Parenteral administration includes injections such as intranasal administration, subcutaneous injection, intramuscular injection, intravenous injection, suppositories and the like. Alternatively, for oral administration, it can be prepared as a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, cellulose and the like.

The vaccine of the invention is administered in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the immune system, and the required dosage will be determined at the discretion of the clinician. Usually, a suitable dose is 1 to 100 mg of CTL epitope peptide per patient, and 10 ⁶ to 10 ⁹ in CTL epitope peptide-pulsed cells. Also, the administration interval can be set according to the subject and purpose.

[Passive immunity vaccine]
The CTL epitope peptides of the present invention can be used for the preparation of passive immunotherapeutic agents. The CTL specific for EBV LMP2 or CTL specific for EBV EBNA1 obtained as described below can be suspended in human albumin-containing PBS or the like to make a passive immunotherapeutic agent for EBV. The EBV-specific CTL contained in the passive immunotherapeutic agent can be obtained by the following preparation method, and can also be purified and used to enhance the purity of the CTL.

CTL preparation method 1
The PBMCs are reacted with an appropriate concentration of EBV-specific MHC-tetramer reagent. Since EBV-specific CTL bound to the MHC-tetramer reagent are stained with the labeling dye, only the stained CTL is isolated using a cell sorter, microscope or the like. The EBV-specific CTL isolated in this manner are T-cell stimulants such as anti-CD3 antibody, PHA, and IL-2 or antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Stimulate and proliferate and secure the number of cells necessary for passive immunotherapy.

CTL preparation method 2
The EBV-specific MHC-monomer and / or MHC-tetramer reagent is immobilized on a sterile plate or the like, and the PBMCs are cultured on the immobilized plate. In order to isolate EBV-specific CTL bound to MHC-monomer and / or MHC-tetramer reagent immobilized on the plate, after washing away other cells suspended without binding, the plate is plated. Suspend only the specific CTL remaining in in fresh medium. Thus, EBV-specific CTL isolated in this manner are stimulated with T-cell stimulants such as anti-CD3 antibody, PHA, and IL-2 or antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Grow and secure the number of cells needed for passive immunotherapy.

CTL preparation method 3
Actogenically against EBV-specific MHC-monomer and / or MHC-tetramer reagents and costimulatory molecules such as CD80, CD83, CD86, or CD28, a ligand on the T cell side that binds costimulatory molecules The antibody and the like are immobilized on a sterile plate and the like, and PBMC are cultured on the immobilized plate. After 2 days, IL-2 is added to the medium and cultured in a 5% CO ₂ thermostat at 37 ° C. for 7 to 14 days. The cultured cells are collected and culture is continued on a new immobilized plate. By repeating this operation, CTLs of the number of cells necessary for passive immunotherapy are secured.

CTL preparation method 4
Stimulation of PBMC or T cells directly with the CTL epitope peptide of the present invention, antigen-presenting cells pulsed with the peptide, antigen-presented cells transfected, or artificial antigen-presenting cells having artificially prepared antigen-presenting ability Do. The stimulation can be in vitro but may be in vivo. When stimulated in vitro, the CTL induced by stimulation are cultured in a 5% CO ₂ thermostat at 37 ° C. for 7 to 14 days. Stimulation with CTL epitope peptide and IL-2 or antigen-presenting cells and IL-2 in culture is repeated once a week to secure CTLs of the number of cells necessary for passive immunotherapy.

Purification method of CTL
In the CTL preparation method, when the proportion of specific CTLs is low, it is possible to recover specific CTLs with high purity by using the following methods as needed.

Purification by MHC-tetramer reagent
Reacting EBV-specific MHC-tetramer reagent with CTL induced by the CTL preparation method, and separating an antibody against a labeling dye labeling the MHC-tetramer reagent using a magnetically labeled secondary antibody It is possible. Such magnetically labeled secondary antibodies and magnetically labeled cell separation devices are available, for example, from Dynal or Miltenyi Biotec GmbH. The EBV-specific CTLs thus isolated are proliferated with T cell stimulating agents such as anti-CD3 antibody, PHA, and IL-2 to secure the number of cells necessary for passive immunotherapy.

Purification by secreted cytokines
EBV-specific CTL can be purified using a cytokine or the like released by EBV-specific CTL. For example, by using a kit available from Miltenyi Biotec GmbH, cytokines released from CTL can be captured by a specific antibody on the cell surface, stained with an anti-cytokine labeled antibody, and subsequently labeled substance specific to a magnetic label After reaction with the antibody, purification using a magnetically labeled cell separation device is also possible. The EBV-specific CTLs thus isolated are proliferated with T cell stimulating agents such as anti-CD3 antibody, PHA, and IL-2 to secure the number of cells necessary for passive immunotherapy.

Purification using cell surface protein specific antibodies Cell surface proteins (eg, CD137, CD107a, CD107b, CD63, CD69, etc.) whose expression is enhanced by specific stimulation have been reported on the cell surface of specific CTL (Betts MR Sensitive and identifiable identification of antigen-specific CD8 + T cells by a flow cytometric assay for degradation. J Immunol Methods. 2003; 281: 65: 78, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA. , Trimble LA, Shankar P, Patterson M, Daily JP, Lieberman J. Human immunodeficiency virus-specific circulating CD8 Lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules for T-cell activation. J Virol. 2000; 74: 7320 -7330). By magnetically labeling a specific antibody of such a protein, it is possible to purify CTL using a magnetic separation device or the like. Also, CTL can be similarly purified by magnetically labeling an anti-IgG antibody or the like to such a specific antibody. Alternatively, these specific antibodies can be coated on a culture plastic plate, the stimulated PBMCs can be cultured using this plate, and the cell population that did not bind to the plate can be washed away to purify specific CTL. It is. The EBV-specific CTLs thus isolated are proliferated with T cell stimulating agents such as anti-CD3 antibody, PHA, and IL-2 to secure the number of cells necessary for passive immunotherapy.

[Quantification of EBV-specific CTL]
It is important information to predict EBV-specific CTL epitope peptides whether EBV-specific CTL is present in peripheral blood of cancer patients or the variation in the amount thereof. The quantification of EBV-specific CTL can be performed by the following three methods using the CTL epitope peptide of the present invention.

Quantitative method 1 (MHC-tetramer method)
The MHC-tetramer reagent produced using the CTL epitope peptide of the present invention can be used to quantify CTL specific for EBV in peripheral blood. The quantification can be performed, for example, as follows. Peripheral blood or PBMCs are reacted with an appropriate concentration of MHC-tetramer reagent. Since CTLs bound to the MHC-tetramer reagent are stained with a labeling dye, they are counted using a flow cytometer, a microscope or the like. When reacting with an MHC-tetramer reagent, the T cell subset of EBV-specific CTL can also be determined simultaneously by reacting an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, etc. labeled with a dye different from the MHC-tetramer reagent. it can.

Quantitative method 2
It is a method for quantifying IFNγ (interferon gamma), TNFα (tumor necrosis factor alpha), cytokines such as interleukins and / or chemokines produced by CTL by stimulating PBMC with the CTL epitope peptide of the present invention. The method is specifically described below taking IFNγ as an example.

2-1 Method 1 (cytokine IFNγ producing cell assay) by cytokine assay
The PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 × 10 ⁶ / mL and the CTL epitope peptide of the invention is added. Furthermore, an intracellular protein transport inhibitor (for example, Brefeldin A, Monensin etc.) is added, and the cells are cultured in a 5% CO ₂ thermostat at 37 ° C. for 5 to 16 hours. After culture, the cells are reacted with a T cell marker antibody (anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody) or an MHC-tetramer reagent, fixed to cells, permeabilized, and reacted with a dye-labeled anti-IFNγ antibody. It analyzes using a flow cytometer etc., and quantifies the percentage of IFNγ positive cells in all cells, in T cells or in MHC-tetramer reagent positive cells.

Method 2 (Erispot assay) by 2-2 cytokine quantification
Apply PBMC to a 96-well MultiScreen-HA plate (Millipore) immobilized with anti-IFNγ antibody. Thereafter, the CTL epitope peptide is placed in each well and cultured in a 5% CO ₂ incubator at 37 ° C. for 20 hours. The next day, the plate is washed and reacted in the order of anti-IFNγ antibody and peroxidase-labeled anti-IgG antibody. Further, a substrate for peroxidase is added, and the IFNγ spot is visualized by color development, and quantified by counting using a stereomicroscope or an ELISPOT analyzer (CTL).

2-3 Method 3 by cytokine quantification (method to quantify IFNγ secreted in culture supernatant)
The PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 × 10 ⁶ / mL and the CTL epitope peptide of the invention is added. Incubate for 24 to 48 hours at 37 ° C. in a 5% CO ₂ thermostat. After culture, the supernatant is collected, and the IFNγ concentration contained therein is quantified using a commercially available ELISA kit (eg, Quantikine ELISA Human IFNγ Immunoassay from R & D Systems).

Quantitative method 3
The quantification is performed using cell surface protein specific antibodies. It has been reported that CTL specific for CTL epitope peptides 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 or the like with a labeled antibody that specifically recognizes a cell surface protein, CTL binds to the labeled antibody and is stained by the labeling dye. The 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 the specific CTL can be determined simultaneously.

[CTL large-scale culture using culture bag]
This culture process does not use dendritic cells for induction of antigen-specific cytotoxic T cells (CTL), which is a conventional method, and can use autologous plasma to prepare a large amount of CTL at low cost and in a simple operation. It is characterized by things. The details of the technology are described in detail in the inventors' patent application (Patent Application No. 2009-166630).

20-200 mL of HLA-A11 positive healthy adult peripheral blood is collected into a heparin collection tube and centrifuged at 3,000 rpm for 10 minutes at room temperature. The supernatant plasma is aliquoted and inactivated (56 ° C., 20 minutes). After centrifuging at 3,000 rpm for 10 minutes at room temperature again, the supernatant is recovered, aliquoted, stored at -30.degree. C., dissolved at any time, and used as culture plasma. The supernatant from which the plasma was separated was added an equal volume of RPMI 1640 medium to isolate PBMC by Ficoll specific gravity centrifugation. The number of cells is counted, 10/11 cells are suspended in RPMI 1640 medium, and culture plasma is added to a final concentration of 10%. Subsequently, add CTL epitope peptide, inject it into a culture bag (CultiLife 215 TAKARA BIO) using a lure lock syringe, and let it stand in a CO ₂ thermostatic bath at 37 ° C. and 5% CO ₂ (CTL induction bag) . 1/11 volume of cells are suspended in RPMI 1640 medium, culture plasma is added to a final concentration of 10%, an anti-CD3 antibody is added to a final concentration of 1 μg / mL, and then using a lure lock syringe It injected 37 ° C. in the culture bag, to stand at CO ₂ thermostat at 5% CO ₂ (antigen-presenting cells induced bag). Two days later, an equal volume of IL-2 containing medium (RPMI 1640 IL-2 100 IU / mL) is injected into the culture bag. After 5 days of culture in a CO ₂ thermostat, an equal volume of IL-2 containing medium (AlyS505N IL-2 100 IU / mL) is injected into the culture bag. Thereafter, every 3 days, an equal volume of IL-2 containing medium (AlyS505N IL-2 100 IU / mL) is injected (the intervals here do not necessarily have to be the same). By the above operation, both antigen-specific CTL and antigen-presenting cells can be induced in about 14 days from the start of culture. After approximately 14 days, cells in the antigen-presenting cell induction bag are collected, counted, centrifuged, suspended in RPMI 1640 medium, added with a CTL epitope peptide, and cultured at room temperature for 1 hour. After centrifugation, the supernatant is aspirated and suspended in IL-2-containing medium (AlyS505N IL-2 100 IU / mL 10% plasma) (peptide-pulsed antigen-presenting cells). Similarly, the cells in the CTL induction bag are collected, the cells are counted, and after centrifugation, the supernatant is aspirated and suspended in an IL-2-containing medium (AlyS505N IL-2 100 IU / mL 10% plasma). The peptide pulse antigen-presenting cells, which are equal in number to the number of cells in the CTL induction bag, are mixed and injected into the culture bag using a lure lock syringe. Thereafter, the culture bag is transferred to a 37 ° C., 5% CO ₂ CO ₂ thermostat to start culture. One day later, growth media (AlyS505N IL-2 1000 IU / mL) is added to the culture bag. After culturing for 3 days in a CO ₂ thermostat, growth medium is added to the culture bag. Thereafter, growth medium is added every two days. By the above operation, antigen-specific CTL can be obtained in a short time of about 20 to 24 days from the start of culture.

[Selection of CTL Epitope Candidate Peptides]
1. Computer-aided analysis The CKAP4-specific CTL epitope candidate peptide of the present invention is a peptide consisting of 8 to 12 amino acids having a binding motif for the desired HLA class I molecule, with respect to the amino acid sequence of CKAP4 protein. Multiple software that can be searched on the Internet (Lundegaard C, Lund O, Buus S, Nielsen M, Major histocompatibility complex class I binding predictions as a tool in epitopic discovery. Immunology., 2010; 130: 309- 318) can be selected and matched.

2. Examination using anchor motif
The HLA class I molecules are mainly HLA-A, HLA-B, and HLA-C, and the epitope peptide presented upon binding to these consists of 8 to 10 amino acids. The 2nd and the 9th or 10th amino acids from the N-terminal side of the epitope peptide are the most important amino acids for binding to HLA class I molecules, and are called anchor motifs. It is reported that this anchor motif is different depending on the type of each HLA class I molecule. For example, as a peptide that binds to the world's most studied HLA-A2 molecule, Leu is located at the second position from the N-terminus, and Leu or Val is located at the 9th or 10th position. The best known peptide is a peptide consisting of 9 to 10 amino acids (T Sudo, N Kamikawaji, A Kimura, Y Date, CJ Savoie, H Nakashima, E Furuichi, S Kuhara, and T Sasazuki Differences in MHC class I self peptide repertoires among HLA-A2 subtypes. J. Immunol., 1995; 155: 4749-4756). In addition, as a peptide that binds to HLA-A24 molecules frequently found in Asian races including Japanese, any of Tyr, Phe, Met or Trp is placed at the second position from the N-terminus, and the 9th or 10th A peptide consisting of 9 to 10 amino acids, in which any of Leu, Ile, Trp or Phe is arranged at the position, is most well known (A Kondo, J Sidney, S Southwood, MF del Guercio , E Appella, H Sakamoto, E Celis, HM Grey, RW Chesnut, and RT Kubo, Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules., J. Immunol., 1995; 155: 4307- 4312). From the amino acid sequence of the protein, a sequence consisting of 8 to 12 amino acids having this anchor motif can be searched to select a CTL epitope candidate peptide.

[Synthesis of peptide]
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. For example, 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. In addition, synthesis by a commercially available chemical synthesizer (for example, peptide synthesizer from Applied Biosystems) is also possible.

[Study of CTL epitope candidate peptides]
As described above, by using the prediction software, it is possible to analyze the amino acid sequence constituting the protein in a short time, and to predict the half life of the binding ability or dissociation between the CTL epitope candidate peptide and HLA. However, candidate peptides obtained using such prediction software do not necessarily function in vivo as CTL epitopes.

  The first reason is that these analysis softwares hardly take into account the efficiency of peptide fragment production by protein degradation. CTL epitopes are composed of peptide fragments of 8 to 12 amino acid residues in length, which are produced in cells by various degradation of antigenic proteins. Specifically, the antigen protein is first degraded in the cytoplasm by the proteasome to form the C-terminus of the peptide. After that, it is transported into the endoplasmic reticulum by TAP (transporter associated with antigen processing) molecules, where the N-terminus is formed by the protease ERAP1 to form a peptide fragment of 8 to 12 amino acid residues in length. In the prediction of CTL epitope candidate peptides, the production process and efficiency of peptide fragments in these cells must be taken into consideration, but this can not be accurately predicted at present. Therefore, among candidate peptides obtained using prediction software, those which can not actually constitute a peptide fragment having a length of 8 to 12 amino acid residues in cells are included.

  The second reason is in the process of selective differentiation of T cells. The cancer antigen is a self antigen, and T cells strongly reacting with peptide fragments derived from the self antigen induce apoptosis in the thymus by negative selection and are eliminated. On the other hand, T cells that respond to foreign antigens are selected by positive selection. From such differentiation process of T cells, T cells that react with peptide fragments derived from autoantigens are usually removed in the thymus, and it is thought that only a small portion exists in peripheral blood. In addition, peptide fragments derived from self antigens 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. In other words, among the peptide fragments derived from self antigens derived from HLA presented on the cell membrane surface, only those that CTL recognize are only a part, and most of the CTL epitope candidate peptides by predicted software are not recognized by CTL. It is believed that. That is, it is considered to be non-antigenic.

  For these reasons, it can be said that obtaining a CTL epitope candidate peptide simply by prediction software and identifying a CTL epitope peptide responsible for an immune response are largely different. The inventors have identified a CTL epitope peptide by a method of directly detecting CTL specific to a candidate peptide in vivo using an MHC-tetramer reagent. That is, detection of a specific CTL by an MHC-tetramer reagent from a sample such as peripheral blood means that a candidate peptide-specific immune response is elicited in vivo, and the candidate peptide is a CTL epitope. Indicates that it is a peptide.

As mentioned above, the CTL epitope candidate peptide selected by the above-mentioned method can become a CKAP4 specific CTL epitope peptide for the first time through examination of (1)-(3) shown below.
(1) Examination using cultured cell lines The peptide fragments generated by proteolysis by proteasome are guided by the TAP molecule to the lumen of the endoplasmic reticulum, bind to the complex of HLA class I molecule and β2-microglobulin, and the cell membrane Transported to the surface. The TAP gene deficient cell line deficient in this TAP molecule can not express a peptide fragment, which is a degradation product of an endogenous protein, on the cell membrane surface. In addition, the human lymphoblastoid cell line T2, which is a typical TAP gene-deficient cell line, or an HLA molecule of a cell line (T2-A24) in which an HLA-A24 molecule has been introduced into T2 has expression on the cell membrane surface. Very unstable. However, when bound to an externally supplied peptide, HLA molecules stabilize on the cell membrane surface. Using this property, TAP gene-deficient cell lines can be used in experiments to verify the binding between HLA molecules and externally supplied peptides. Specifically, after mixed culture of a TAP gene-deficient cell line and a CTL epitope candidate peptide, the cells are stained with an anti-HLA antibody, and the change in the expression intensity of the HLA molecule is calculated by flow cytometry to obtain the target HLA molecule The binding properties of CTL epitope candidate peptides can be examined. When a CTL epitope candidate peptide is bound to an HLA molecule expressed by a TAP gene-deficient cell line, the complex of the HLA molecule and the peptide is stabilized on the cell membrane surface, and when stained with an anti-HLA antibody, expression of the HLA molecule is enhanced Is observed. On the other hand, when the added CTL epitope candidate peptide does not show binding to the HLA molecule, the HLA molecule on the cell membrane surface is unstable, and even if it is stained with the anti-HLA antibody, the enhanced expression of the HLA molecule is not confirmed. Using such a method, it is possible to verify the binding between an HLA molecule and a CTL epitope candidate peptide.
(2) Folding test
The MHC-tetramer reagent is a reagent in which a three-way complex (MHC-monomer) of MHC (HLA in case of human) and β2-microglobulin and a peptide fragment is produced in a test tube, and the reagent is obtained by tetramerizing the MHC-monomer. is there. The MHC-tetramer reagent is the only reagent that can selectively detect antigen-specific CTLs that show MHC restriction. In addition, the MHC-tetramer reagent can not only be able to quantify the amount of CTL by analyzing with flow cytometry after co-staining with anti-CD (cluster of differentiation) antibody or anti-cytokine antibody etc., but also its activation state or differentiation stage. It is possible to evaluate every single cell. The first step in the preparation of the MHC-tetramer reagent begins by folding the raw materials MHC, β2-microglobulin and peptide in a suitable solution in a test tube. In the folding solution, a three-way complex (MHC-monomer) is formed by the association reaction of the three raw materials. At this time, if the binding strength between the MHC and the peptide is high, the association reaction proceeds smoothly, and analysis with a gel filtration column makes it possible to detect a complex of three types of raw materials (MHC-monomer). On the other hand, when there is no binding ability between MHC and peptide, MHC-monomer is hardly detected. Therefore, it is possible to verify the binding and stability of the MHC and the peptide by analyzing the folding solution with time or by performing a heat treatment or the like.
(3) Detection of Epitope Peptide-Specific CTLs Peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals are suspended in an appropriate medium at a cell concentration of 1 to 3 × 10 ⁶ / mL. Add any of the synthesized CTL epitope candidate peptides (SEQ ID NOS: 36 to 47) or several kinds thereof to a concentration of 1 to 100 μg / mL, and dispense 100 μL / well to a 96-well culture plate . Incubate at 37 ° C. in a 5% CO ₂ thermostat and after 2 days add IL-2. Thereafter, CTL is induced by stimulation with peptide and IL-2 once every 7 to 14 days. Examination of whether the thus derived CTL has specificity for a CTL epitope candidate peptide is determined by the MHC-tetramer method. When CTLs that can be stained with the MHC-tetramer reagent are detected, the CTL epitope candidate peptide used can be identified as an antigenic peptide (epitope peptide) having CTL inducibility. Generally, T cells (naive T cells) exported from the thymus are activated and differentiated into effector T cells only upon antigen stimulation by antigen presenting cells such as dendritic cells and macrophages. In this method of simply culturing PBMCs and peptides in a mixed culture, the possibility of differentiation of naive T cells in peripheral blood was low and was present in PBMCs, in particular because artificially prepared antigen-presenting cells were not used. It is thought that effector / memory T cells are proliferating by peptide stimulation. Therefore, when CTLs that can be stained with the MHC-tetramer reagent are detected, it means that effector / memory type CTLs are originally present in the donor peripheral blood PBMC, and the antigenic peptide is constantly determined. It suggests that the mediated immune response is elicited in vivo.

[Production of CKAP 4 Specific MHC-Monomer and MHC-Tetramer Reagent]
MHC-monomer and MHC-tetramer reagents using CKAP4-specific CTL epitope candidate peptides can be prepared by known methods (US Patent Number 5, 635, 363, French Application Number FR9911133). An MHC-monomer, which is a complex of three HLA class I molecules purified from a recombinant host for protein expression, β2-microglobulin and the CKAP4 specific CTL epitope candidate peptide of the present invention, is formed in a folding solution. A biotin binding site is previously added to the C terminus of the recombinant HLA class I molecule, and biotin is added to this site after MHC-monomer formation. The MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4. Moreover, the differentiation stage of CTL can be examined by using it in combination with antibodies against cell surface proteins (such as CD62L, CCR7 and CD45RA) (Seder RA, Ahmed R. Similarities and differences in CD4 + and CD8 + effector and memory T cell generation Nat Immunol. 2003; 4: 835-842). Alternatively, it can be used for functional evaluation of CTL in combination with intracellular cytokine staining. For example, in hepatitis C, CTL against HCV (Hepatitis C virus) exists as one of the causes of sustained infection, but CTL does not produce cytokines or the like, or the proportion of CTL produced is extremely low. It has been reported that it may be chemically unresponsive (anergy) (Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, Walker B, Sullivan J, Phillips R, Pape GR Klerman P. Sustained dysfunction of antiviral CD8 + T lymphocytes after infection with hepatitis C virus. J Virol. 2001; 75: 5550-5558). In addition, for HCMV-specific CTL after bone marrow transplantation, it is thought that not only examining the presence or absence of the CTL but examining the strength of cytokine production ability is effective for measuring the timing of antiviral drug administration etc. (Ozdemir E, St John LS, Gillespie G, Rowland-Jones S, Champlin RE, Molldrem JJ, Komanduri KV. Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8. 2002; 100: 3690-3697). By identifying such a specific CTL epitope peptide and manufacturing an MHC-tetramer reagent, it becomes possible to quantify and characterize specific CTL, and a large contribution can be made in obtaining diagnostic information.

[Active immune vaccine]
Peptide Vaccines The CTL epitope peptides of the invention can be used as peptide vaccines in active immunotherapy. That is, a vaccine comprising the CTL epitope peptide of the present invention is administered to a patient, CKAP4-specific CTL can be expanded in the body, and treatment for malignant tumors 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. The antigen-presenting cell on which the CTL epitope peptide is presented is
1. CTL epitope peptide-pulsed antigen-presenting cells in which antigen-presenting cells and CTL epitope peptide are mixed for 30 minutes to 1 hour in a suitable culture solution. 2. Cells in which an antigen-presenting cell is presented with a CTL epitope peptide by gene transfer or the like using a nucleic acid encoding the CTL epitope peptide. It means an artificial antigen presenting cell having an antigen presenting ability prepared artificially. The antigen-presenting cells include, for example, dendritic cells, B cells, macrophages, certain T cells, etc., but are cells expressing an HLA molecule to which the peptide can be bound on the cell membrane surface, such as CTLs. It means one having stimulation ability. An artificially prepared antigen-displaying cell having an antigen-displaying ability is prepared, for example, by immobilizing a ternary complex of an HLA molecule, a CTL epitope peptide and β2-microglobulin on a bead such as a lipid bilayer membrane, plastic or latex. Or a costimulatory molecule such as CD80, CD83 or CD86 that can stimulate CTL, or fix an antibody that acts on an agonistic effect on CD28, which is a T cell side ligand that binds to the costimulatory molecule. (Oelke M, Maus MV, Didiano D, June CH, Mackensen A, Schneck JP. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med. 2003; 9: 619-624, Walter S, Herrgen L, Schoor O, Jung G, Wernet D, Buhring HJ, Rammensee HG, Stevanovic S. 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-presinging constructs efficient histocompatibility minor-histocompatibility antigen-specific cytotoxic T lymphocytes; -226).

Genetic Vaccine The nucleic acid of the CTL epitope peptide of the present invention can be used in DNA vaccines, recombinant viral vector vaccines and the like in active immunotherapy. In this case, it is desirable to change the nucleic acid sequence of the CTL epitope peptide to a recombinant vaccine or codon usage compatible with the host producing the recombinant virus vaccine (Casimiro, DR et al. Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid J. Virol., 2003; 77: 6305-6313, Berzofsky JA, Ahlers JD, Janik J, Morris J, Oh S, Terabebe, Recombinational Vaccinia Virus, and Replication-Defective Adenovirus Vectors M, Belyakov IM. Progress on new vaccine strategies against chronic viral infections. J Clin Invest. 2004; 114: 450-462).

  Vaccines comprising the CTL epitope peptides of the present invention, or antigen presenting cells on which CTL epitope peptides are presented can be prepared using methods known in the art. For example, such 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 the form of a neutral or salt, for example, pharmaceutically acceptable salts include inorganic salts such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid and tartaric acid . In addition, antigen-presenting cells on which the CTL epitope peptide of the present invention is presented are pharmaceutically acceptable and an excipient compatible with the peptide or the activity of the cells, such as water, saline, dextrose, ethanol, It can be used by mixing with glycerol, DMSO (dimethyl sulfoxide), and other adjuvants or the like, or a combination thereof. Furthermore, if necessary, adjuvants such as albumin, a wetting agent and an emulsifying agent may be added.

  The vaccine of the present invention can be administered by parenteral administration and oral administration, but parenteral administration is generally preferred. Parenteral administration includes injections such as nasal administration, subcutaneous and intradermal injection, intramuscular injection, intravenous injection, suppositories and the like. Alternatively, for oral administration, it can be prepared as a mixture with excipients such as starch, mannitol, lactose, magnesium stearate, cellulose and the like.

The vaccine of the invention is administered in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the immune system, and the required dosage will be determined at the discretion of the clinician. Usually, a suitable dose is 1 to 100 mg of CTL epitope peptide per patient, and 10 ⁶ to 10 ⁹ in CTL epitope peptide-pulsed cells. Also, the administration interval can be set according to the subject and purpose.

[Passive immunity vaccine]
The CTL epitope peptides of the present invention can be used for the preparation of passive immunotherapeutic agents. The CTL specific for CKAP4 obtained as described below can be suspended in human albumin-containing PBS or the like to make a passive immunotherapeutic agent for malignant tumors expressing CKAP4. The CKAP 4 specific CTL 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 the CTL.

CTL preparation method 1
The PBMCs are reacted with an appropriate concentration of CKAP4-specific MHC-tetramer reagent. Since CKAP4 specific CTLs bound to the MHC-tetramer reagent are stained by the labeling dye, only the stained CTLs are isolated using a cell sorter, a microscope or the like. The CKAP4-specific CTL isolated in this manner are T-cell stimulants such as anti-CD3 antibodies, PHA and IL-2, and antigen-presenting cells whose proliferation ability has been lost by X-ray irradiation or mitomycin treatment. Stimulate and proliferate and secure the number of cells necessary for passive immunotherapy.

CTL preparation method 2
CKAP4-specific MHC-monomer and / or MHC-tetramer reagent is immobilized on a sterile plate or the like, and PBMCs are cultured on the immobilized plate. In order to isolate CKAP4-specific CTL bound to MHC-monomer and / or MHC-tetramer reagent immobilized on the plate, after washing away other cells suspended without binding, the plate is plated. The remaining CKAP4 specific CTLs are suspended in fresh medium. The CKAP4-specific CTL thus isolated can be stimulated and proliferated by T-cell stimulants such as anti-CD3 antibody, PHA and IL-2, and antigen-presenting cells whose proliferation ability has been lost by X irradiation or mitomycin treatment. To secure the number of cells required for passive immunotherapy.

CTL preparation method 3
It acts as an agonistic agent on CKAP4-specific MHC-monomer and / or MHC-tetramer reagents and costimulatory molecules such as CD80, CD83, and CD86, or CD28, which is a T cell side ligand that binds to costimulatory molecules. The antibody and the like are immobilized on a sterile plate and the like, and PBMC are cultured on the immobilized plate. Two days later, IL-2 is added to the medium and cultured in a 5% CO ₂ thermostat at 37 ° C. for 7 to 10 days. The cultured cells are collected and culture is continued on a new immobilized plate. By repeating this operation, CTLs of the number of cells necessary for passive immunotherapy are secured.

CTL preparation method 4
Stimulation of PBMC or T cells directly with the CTL epitope peptide of the present invention, antigen-presenting cells pulsed with the peptide, antigen-presented cells transfected, or artificial antigen-presenting cells having artificially prepared antigen-presenting ability Do. The stimulation can be in vitro but may be in vivo. When stimulated in vitro, the CTL induced by stimulation are cultured in a 5% CO ₂ thermostat at 37 ° C. for 7 to 14 days. Stimulation with CTL epitope peptide and IL-2 or antigen-presenting cells and IL-2 in culture is repeated once a week to secure CTL of the number of cells necessary for passive immunotherapy.

Purification method of CTL
In the CTL preparation method, when the ratio of specific CTL is low, specific CTL can be recovered with high purity by using the following method as needed.

Purification by MHC-tetramer reagent
It is possible to react CKAP4-specific MHC-tetramer reagent with CTL induced by CTL preparation method, and separate the antibody against the labeling dye labeling the MHC-tetramer reagent using the second antibody magnetically labeled. It is possible. Such magnetically labeled secondary antibodies and magnetically labeled cell separation devices are available, for example, from Dynal or Miltenyi Biotec GmbH. The CKAP4-specific CTL thus isolated are stimulated and proliferated with T cell stimulating agents such as anti-CD3 antibody, PHA, and IL-2 to secure the number of cells necessary for passive immunotherapy.

Purification by secreted cytokines
CKAP4-specific CTL can be purified using a cytokine or the like released by CKAP4-specific CTL. For example, by using a kit available from Miltenyi Biotec GmbH, a cytokine released from CTL is complemented by a specific antibody on the cell surface, stained with a cytokine specific labeled antibody, and then magnetically labeled labeled substance specific It is also possible to use the magnetically labeled cell separator to purify after reacting with the relevant antibodies. The CKAP4-specific CTL thus isolated are stimulated and proliferated with T cell stimulating agents such as anti-CD3 antibody, PHA, and IL-2 to secure the number of cells necessary for passive immunotherapy.

Purification using cell surface protein specific antibodies Cell surface proteins (eg, CD137, CD107a, CD107b, CD63, CD69, etc.) whose expression is enhanced by specific stimulation have been reported on the cell surface of specific CTL (Betts MR Sensitive and identifiable identification of antigen-specific CD8 + T cells by a flow cytometric assay for degradation. J Immunol Methods. 2003; 281: 65: 78, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M. , Trimble LA, Shankar P, Patterson M, Daily JP, Lieberman J. Human immunodeficiency virus-specific circulating CD8 Lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules for T-cell activation. J Virol. 2000; 74: 7320 Int J Hematol-7330, Watanabe K, Suzuki S, Kamei M, Toji S, Kawase T, Takahashi T, Kuzushima K, and Akatsuka Y. CD 137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adaptive immunotherapy. , 2008; 88; 311-320). By magnetically labeling a specific antibody against such a protein, it is possible to purify CKAP4 specific CTL using a magnetic separation device or the like. In addition, the CKAP4-specific CTL can be similarly purified by magnetically labeling an anti-IgG antibody or the like to such a specific antibody. Alternatively, these specific antibodies may be coated on a culture plastic plate, the stimulated PBMCs may be cultured using this plate, and the CKAP4-specific CTL may be purified by washing out the non-plate-bound cell population. It is possible. The CKAP4-specific CTL thus isolated are stimulated and proliferated with T cell stimulating agents such as anti-CD3 antibody, PHA, and IL-2 to secure the number of cells necessary for passive immunotherapy.

[Quantification of CKAP4 specific CTL]
Knowing whether CKAP4-specific CTL is present in the peripheral blood of cancer patients or the variation in the amount predicts the efficacy of active and passive immune vaccines utilizing CKAP4-specific CTL epitope peptides It is important information for In addition, in the case of a passive immune vaccine, it is necessary to measure in advance the content of CKAP4 specific CTL in the preparation. The quantification of CKAP4 specific CTL can be performed by the following three methods using the CTL epitope peptide of the present invention.

Quantitative method 1 (MHC-tetramer method)
The MHC-tetramer reagent produced using the CTL epitope peptide of the present invention can be used to quantify CTL specific for CKAP4 in peripheral blood. The quantification can be performed, for example, as follows. Peripheral blood or PBMCs are reacted with an appropriate concentration of MHC-tetramer reagent. Since CTLs bound to the MHC-tetramer reagent are stained with a labeling dye, they are counted using a flow cytometer, a microscope or the like. When reacting with an MHC-tetramer reagent, the T cell subset of CKAP4 specific CTL is also determined simultaneously by reacting an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, etc. labeled with a dye different from the MHC-tetramer reagent. it can.

Quantitative method 2
It is a method for quantifying IFNγ (interferon gamma), TNFα (tumor necrosis factor alpha), cytokines such as interleukins and / or chemokines produced by CTL by stimulating PBMC with the CTL epitope peptide of the present invention. The method is specifically described below taking IFNγ as an example.

2-1 Method 1 (cytokine IFNγ producing cell assay) by cytokine assay
The PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 × 10 ⁶ / mL and the CTL epitope peptide of the invention is added. Furthermore, an intracellular protein transport inhibitor (for example, Brefeldin A, Monensin etc.) is added, and the cells are cultured in a 5% CO ₂ thermostat at 37 ° C. for 5 to 16 hours. After culture, the cells are reacted with a T cell marker antibody (anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody) or an MHC-tetramer reagent, fixed to cells, permeabilized, and reacted with a dye-labeled anti-IFNγ antibody. It analyzes using a flow cytometer etc., and quantifies the percentage of IFNγ positive cells in all cells, in T cells or in MHC-tetramer reagent positive cells.

Method 2 (Erispot assay) by 2-2 cytokine quantification
Apply PBMC to a 96-well MultiScreen-HA plate (Millipore) immobilized with anti-IFNγ antibody. Thereafter, the CTL epitope peptide is placed in each well and cultured in a 5% CO ₂ incubator at 37 ° C. for 20 hours. The next day, the plate is washed and reacted in the order of anti-IFNγ antibody and peroxidase-labeled anti-IgG antibody. Further, a substrate for peroxidase is added, and the IFNγ spot is visualized by color development, and quantified by counting using a stereomicroscope or an ELISPOT analyzer (CTL).

2-3 Method 3 by cytokine quantification (method to quantify IFNγ secreted in culture supernatant)
The PBMCs are suspended in a suitable medium at a cell concentration of approximately 2 × 10 ⁶ / mL and the CTL epitope peptide of the invention is added. Incubate for 24 to 48 hours at 37 ° C. in a 5% CO ₂ thermostat. After culture, the supernatant is collected, and the IFNγ concentration contained therein is quantified using a commercially available ELISA kit (eg, Quantikine ELISA Human IFNγ Immunoassay from R & D Systems).

Quantitative method 3
The quantification is performed using cell surface protein specific antibodies. CTL specific for CTL epitope peptide has been reported to be enhanced in 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. CD 137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adaptive immunotherapy. Int J Hematol, 2008; 88; 311-320). Therefore, by mixing PBMC stimulated with a CTL epitope peptide or the like and a labeled antibody that specifically recognizes a cell surface protein, CTL binds to the labeled antibody and is stained by the labeling dye. The 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, a T cell subset of specific CTL can be determined simultaneously.

[Antibody specific for peptide-MHC complex]
The identified monoclonal antibody (hereinafter pMHC antibody) specific to the complex of the cancer antigen epitope peptide and the MHC can specifically detect cancer cells presenting the epitope peptide on the cell membrane surface. Therefore, pMHC antibodies can be used not only as diagnostic agents for cancer immunotherapy, but also as therapeutic antibodies with high specificity by combining antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC activity) and anticancer agents. It is useful. The preparation of pMHC antibodies is generally performed by phage display. The phage display method is a system for expressing a foreign gene as a fusion protein so as not to lose the infectivity of phage. It is known that specific monoclonal antibodies can be isolated by expressing and screening antibody variable regions in phage using this (Tsukahara T, Emori M, Murata K, Hirano T, Muroi N, Kyono M, Toji S, Watanabe K, Torigoe T, Kochin V, Asanuma H, Matsumiya H, Yamashita K, Himi T, Ichimiya S, Wada T, Yamashita T, Hasegawa T, Sato N. Specific targeting of a naturally presented osteosarcoma antigen, papillomavirus binding factor peptide, using an artificial monoclonal antibody. J Biol Chem. 2014; 289 (32): 22035-22047). Acquisition of pMHC antibodies is performed in the flow of preparation of antibody library, panning, isolation of antibodies, evaluation. For panning, a complex of a cancer antigen epitope peptide and MHC (MHC-monomer) is immobilized on an ELISA plate etc. or immobilized by biotin-avidin binding, and this is reacted with a phage library. By repeating the washing and elution, a highly binding antibody specific to the epitope peptide-MHC complex can be isolated. 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.

  All prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES The present invention will be specifically described below by way of Examples, but the present invention is not limited thereto. Unless otherwise noted, the experimental method was carried out with reference to the written book (Immunological Experiment Operating Method, Shunsuke Akita, Susumu Shibata, Honjo, Toshiyuki Tsujioka, Minami-Edo 1995).
Example 1
[Selection of EBV-Specific CTL Epitope Candidate Peptides]
EBV is classified into type 1 and type 2 according to the difference in expressed protein and the like. Most are type 1 infections / latencies in humans, and type 2 infections including AG 876 are rare in Asia (Dambaugh T, Hennessy K, Chamnankit L, Kieff E. U2 region of Epstein- Barr virus DNA may encode Epstein-Barr nuclear antigen 2. Proc Natl Acad Sci US A. 1984 Dec; 81 (23): 7632-6). However, cases of EBV co-infection in the same individual have also been reported, and racial- and region-dependent diversity of EBV-infected strains has been reported (Apolloni A, Sculley TB. Detection of A-type and B- Virology. 1994 Aug 1; 202 (2): 978-81, Correa RM, Fellner MD, Alonio LV, Durand K, Teyssie AR, Picconi MA. Epstein-barr virus (EBV) type Epstein-Barr virus in throat washings and lymphocytes. ) In healthy carriers: Distribution of genotypes and 30 bp deletion in late membrane protein-1 (LMP-1) oncogene. J Med Virol. 2004 Aug; 73 (4): 583-8, Klemenc P, Marin J, Soba E, Gale N, Koren S, Strojan P. Distribution of Epstein-Barr virus genotypes in throat washings, sera, peripheral blood lymphocytes and in EBV positive tumors biopsies from patients with nasopharyngeal carcinoma. Association between Epstein-Barr virus and Hodgkin's lymphoma in Belgium: a pathological and virological study. Leuk Lyk 1083-90, Trimeche M, Bonnet C, Korbi S, Boniver J, de Leval L. 2007 Jul; 48 (7): 1323-31, Tiwawech D, Srivatanakul P, Karalak A, Ishida T. Association between EBNA2 and LMP1 subtypes of Epstein-Barr virus and nasopharyngeal carcinoma in Thais.
J Clin Virol. 2008 May; 42 (1): 1-6.). So far, multiple EBV strains have been isolated, but the problem of co-infection and strain diversity has made it difficult to analyze the base sequence, and the entire base sequence was determined as B95.8, AG876. (MU), MUTU, GD1, GD2, HKNPC1 and AKATA in total 7 only (Baer R, Bankier AT, Biggin MD, Deininger PL, Farrell PJ, Gibson TJ, Hatfull G, Hudson GS, Satchwell SC, Seguin C, Nature. 1984 Jul 19-25; 310 (5974): 207-11, Zeng MS, Li DJ, Liu QL, Song LB, Li MZ, et al., et al., DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Genomic sequence analysis of Epstein-Barr virus strain GD 1 from a nasopharyngeal carcinoma patient. J Virol. 2005 Dec; 79 (24): 15323-30, Dolan A, Addison A, Zhang RH, Yu XJ, Wang HM, Ernberg I C, Gatherer D, Davison AJ, McGeoch DJ. The genome of Epstein-Barr virus type 2 strain AG876.
Virology. 2006 Jun 20; 350 (1): 164-70, Liu P, Fang X, Feng Z, Guo YM, Peng RJ, Liu T, Huang Z, Feng Y, Sun X, Xiong Z, Guo X, Pang SS Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma, Wang B, Lv X, Feng FT, Li DJ, Chen LZ, Huang WL, Zeng MS, Bei JX, Zhang Y, Zeng YX. J Virol. 2011 Nov; 85 (21): 11291-9., Kwok H, Tong AH, Lin CH, Lok S, Farrell PJ, Kwong DL, Chiang AK. Genomic sequencing and comparable. tissue by using next-generation sequencing technology. PLoS One. 2012; 7 (5): e36939., Lin Z, Wang X, Strong MJ, Concha M, Baddoo M, Xu G, Baribault C, Fewell C. Analysis of Epstein-Barr virus genome isolated from primary nasopharyngeal carcinoma biopsy. Hulme W, Hedges D, Taylor CM, Flemington EK. Whole-genome sequencing of the Akata and Mutu Epstein-Barr virus strains. J Virol. 2013 Jan; 87 (2): 1172-82.

  Differences in the expression of major proteins among the strains, amino acid sequence differences have been partially analyzed, and several reports have been made for LMP2 and EBNA1 (Midgley RS, Bell AI, Yao QY, Croom- Carter D, Hislop AD, Whitney BM, Chan AT, Johnson PJ, Rickinson AB. HLA-A11-restricted epitope polymorphism among the Epstein-Barr virus strains in the highly HLA-A11-positive Chinese population: incidence and immunogenicity of variants sequences. J Virol. 2003 Nov; 77 (21): 11507-16, Wang X, Liu X, Jia Y, Chao Y, Xing X, Wang Y, Luo B. Widespread sequence variation in the Epstein-Barr virus latent membrane protein 2A gene J. Northern China isolates. J Gen Virol. 2010 Oct; 91 (Pt 10): 2564-73., Han J, Chen JN, Zhang ZG, Li HG, Ding YG, Du H, Shao CK. Sequence variations of late membrane protein 2A in Epstein-Barr virus-associated gastric carcinoma from Guangzhou, southern China. PLoS One. 2012; 7 (3): e34276.).

  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), which is the most representative strain belonging to EBV type 1, and other strains. It carried out with reference to the mutation of the amino acid in. Specifically, this was performed by collating with a plurality of software published on the Internet, which can search for CTL epitope candidate peptides consisting of 8 to 10 amino acids having a binding motif for the HLA-A11 molecule. As a result, a total of 33 types of 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 EBV LMP2 and EBNA1 are selected: 22 types for LMP2 and 11 types for EBNA1 These peptides were synthesized and shown below as CTL epitope candidate peptides. Amino acid mutations in different EBV strains are underlined. In the present invention, B95.8 strain is a wild strain, and substitution of amino acids in other strains is called mutation. As a peptide for positive control, HLA-A * 11: 01 restricted epitope peptide (ATVQGQNLK, SEQ ID NO: 34) derived from pp65 protein of human cytomegalovirus (CMV) was synthesized. As a peptide for negative control, an HLA-A * 24: 02 restricted epitope peptide (AYACNTSTL, SEQ ID NO: 35) of survivin-2B was synthesized.

(1) Synthesized EBV LMP2-derived HLA-A * 11: 01 restricted CTL epitope peptide candidate
Ala Ser Ser Tyr Ala Ala Ala Gln Arg Lys (SEQ ID NO: 1)
Ala Asn Ser Tyr Ala Ala Ala Gln Arg Lys (SEQ ID NO: 2)
Ala Ser Ser Ser Ala Ala Ala Gln Arg Lys (SEQ ID NO: 3)
Ala Asn Ser Ser Ala Ala Ala Gln Arg Lys (SEQ ID NO: 4)
Ala Ser Ser Tyr Ala Ala Ala Gln Arg (SEQ ID NO: 5)
Ser Ser Tyr Ala Ala Ala Gln Arg Lys (SEQ ID NO: 6)
Val Met Leu Val Leu Leu Ile Leu Ala Tyr (SEQ ID NO: 7)
Leu Val Leu Leu Ile Ala Tyr Arg (SEQ ID NO: 8)
Leu Val Leu Leu Ile Leu Ala Tyr Arg Arg (SEQ ID NO: 9)
Leu Ala Tyr Arg Arg Arg Trp Arg Arg (SEQ ID NO: 10)
Thr Thr Met Phe Leu Leu Met Leu Leu (SEQ ID NO: 11)
Lys Ile Leu Leu Ala Arg Leu Phe Leu Tyr (SEQ ID NO: 12)
Lys Val Leu Leu Ala Arg Leu Phe Leu Tyr (SEQ ID NO: 13)
Ile Leu Leu Ala Arg Leu Phe Leu Tyr (SEQ ID NO: 14)
Val Leu Leu Ala Arg Leu Phe Leu Tyr (SEQ ID NO: 15)
Gly Ser Ile Leu Gln Thr Asn Phe Lys (SEQ ID NO: 16)
Leu Thr Glu Trp Gly Ser Ser Gly Asn Arg (SEQ ID NO: 17)
Arg Thr Tyr Gly Pro Val Phe Met Cys Leu (SEQ ID NO: 18)
Arg Thr Tyr Gly Pro Val Phe Met Ser Leu (SEQ ID NO: 19)
Thr Val Met Ser Asn Thr Leu Leu Ser (SEQ ID NO: 20)
Thr Val Met Thr Asn Thr Leu Leu Ser (SEQ ID NO: 21)
Ala Leu Phe Gly Val Ile Arg Cys Cys Arg (SEQ ID NO: 22)

(2) Synthesized EBV EBNA1-derived HLA-A * 11: 01-restricted CTL epitope peptide candidate
Gly Pro Gly Asn Gly Leu Gly Glu Lys (SEQ ID NO: 23)
Ser Ser Ser Gly Ser Pro Pro Arg Arg (SEQ ID NO: 24)
Gly Gln Gly Asp Gly Gly Arg Arg Arg (SEQ ID NO: 25)
His Arg Gly Gly Gln Gly Gly Ser Asn Pro Lys (SEQ ID NO: 26)
His Arg Gly Glu Glu Gly Gly Ser Ser Gln Lys (SEQ ID NO: 27)
His Arg Gly Gly Gln Gly Gly Ser Asn Gln Lys (SEQ ID NO: 28)
Gly Val Phe Val Tyr Gly Gly Ser Lys (SEQ ID NO: 29)
Lys Thr Ser Leu Tyr Asn Leu Arg Arg (SEQ ID NO: 30)
Gln Thr His Ile Phe Ala Glu Val Leu Lys (SEQ ID NO: 31)
Gly Arg Gly Arg Gly Arg Arg Gly Glu Lys (SEQ ID NO: 32)
Ala Ile Lys Asp Leu Val Met Thr Lys (SEQ ID NO: 33)

(3) Peptides synthesized for control
Ala Thr Val Gln Gly Gln Asn Leu Lys (SEQ ID NO: 34)
Ala Tyr Ala Cys Asn Thr Ser Thr Leu (SEQ ID NO: 35)

  Tables 1, 2, and 3 show the characteristics of LMP2 and EBNA1 HLA-A11: 01 restricted CTL epitope peptide synthesized and the peptide synthesized for control. The peptide names are indicated by three amino acid sequences from the N-terminal side of the synthesized peptide. From the left, the peptide name (a in the table), the amino acid sequence (b in the table), the position on the amino acid sequence of the derived protein, the number of amino acids, NetMHC 3.4 (http://www.cbs.dtu.dk used for analysis) / services / NetMHC /) HLA Peptide Binding Predictions (Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, Brunak S, Lund O. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations Protein Science Sci. 2003 May; 12 (5): 1007-17, Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M. Net MHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class Nucleic Acids Res. 2008 Jul 1; 36 (Web Server issue): W 509-12., Lundegaard C, Lund O, Nielsen M. Accurate approximation method for prediction of class I MHC affinities for 2008 Jun 1; 24 (11): 1397-8.) The score (c in the table) was calculated using the peptides of lengths 8, 10 and 11 using prediction tools trained on 9 mers. We have shown in the order. This score is a numerical value to predict the affinity between HLA-A11 and the peptide, and the higher the score, it means that the HLA and the 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 by the 11 types of analysis software used for analysis.

[Folding test of EBV-specific CTL epitope candidate peptide]
The inventors performed a folding test using 35 kinds of peptides described in Table 1, Table 2 and Table 3. Specifically, HLA-A * 11: 01 and β2-microglobulin expressed and purified using E. coli expression system, and a synthetic peptide are added to the folding solution and mixed, and then the folding solution is separated over time. The analysis was performed on a gel filtration column. In gel filtration column analysis, when the formation of a ternary complex (MHC-monomer) of HLA-A * 11: 01 and β2-microglobulin and an EBV-specific CTL epitope candidate peptide is observed, the MHC-monomer is from the raw material Because of the large molecular weight, elution time in gel filtration column analysis is faster. Also, 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 in the candidate peptide which does not bind to an HLA molecule. A representative gel filtration column analysis in which MHC-monomer formation is observed is shown in FIG.

  According to the results of gel filtration column analysis, when expressing and purifying HLA molecules and β2-microglobulin using an E. coli expression system, the insoluble fraction is purified as inclusion bodies and then solubilized in 8 M urea, but it is difficult Those HLA molecules that are soluble are detected in 7 to 8 minutes as aggregates that do not lead to MHC-monomer formation. However, most of the aggregates are removed by filter filtration which is a pretreatment step of gel filtration column analysis. β 2 -microglobulin is a soluble protein, is solubilized in the folding solution, and is detected around 14 minutes using Superdex 75 10/300 GL (GE Healthcare). After 15 minutes, the composition or peptide of the folding solution is detected. Although the peak of MHC-monomer is not confirmed immediately after the start of the folding test (day 0), the peak becomes large after 1 day (day 1) and 3 days (day 3), and MHC-monomer formation is progressing smoothly It shows the thing.

  The analysis results after 1, 3, and 7 days of the folding test performed on 35 kinds of peptides are shown in FIG. 2 and FIG. As a positive control peptide, the CMV pp65 protein-derived HLA-A * 11: 01 restricted epitope peptide (ATVQGQNLK, SEQ ID NO: 34), as a negative control, survivin-2B-derived HLA-A * 24: 02 restricted epitope peptide (ATVQGQNLK) AYACNTSTL, SEQ ID NO: 35) was used for comparison. The area of the peak showing MHC-monomer formation is indicated by a bar graph. The results of comparison of SEQ ID NOS: 1 to 22 (LMP2) and SEQ ID NOS: 23 to 33 (EBNA1) with SEQ ID NOS: 34 to 35 as positive control peptide and negative control are shown in Table 4 and Table 5, respectively.

  Tables 4 and 5 show HLA-A * 11: 01 binding of peptide candidates. In the folding test, the presence or absence of binding is indicated as ○ with a binding property above the highest binding value (135, 674 μV * second) of the negative control, and × below the negative control without binding. Thus, the HLA-A * 11: 01 binding determined by the experimental values is not necessarily consistent with the predicted value obtained by the computer algorithm, and the experimental values of the folding test are the candidate peptide and the HLA-A in vitro. * 11 Indicates the bondability with 01:01. The results of the folding test experiments showed that all of the lower (predicted value <0.400) eight peptide candidates predicted by the computer algorithm showed HLA-A * 11: 01 binding. On the other hand, predicted values of four peptides not showing HLA-A * 11: 01 binding in the result of the experiment of the folding test are as follows. VML: 0.417; LVL (9 mer): 0.439; TTM: 0.477; KIL: 0.602. It can be seen that none of the predicted values bind to HLA-A * 11: 01 despite being higher than 0.400. Thus, the computer algorithm alone can not determine the binding between the epitope candidate peptide and the HLA molecule.

[Production of EBV LMP 2 Specific MHC-Tetramer Reagent]
Based on the results of the folding test, PE-labeled MHC-tetramer reagents were produced using HLA-A * 11: 01-binding EBV LMP2-specific or EBV EBNA1-specific CTL epitope candidate peptides. The MHC-tetramer reagent prepared in the present invention is indicated, for example, by the abbreviation ASS (10 mer) -Tet, which comprises HLA-A * 11: 01 and ASS (10 mer) peptide (ASSYAAAQRK, SEQ ID NO: 1) and β2 -Shows what was produced using a ternary complex of microglobulin. MHC-monomer, which is a complex of HLA class I molecule, β2-microglobulin and EBV LMP2 or EBV EBNA1 specific CTL epitope candidate peptide of the present invention, purified from recombinant host for protein expression in appropriate folding solution To form. A biotin binding site is previously added to the C terminus of the recombinant HLA class I molecule, and biotin is added to this site after MHC-monomer formation. The MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4.

[Identification of EBV-specific CTL epitope peptide]
(Selection of HLA type of sample)
As EBV-specific CTL epitope candidate peptides, 33 candidate peptides having a binding motif for HLA-A * 11: 01 were selected. Furthermore, the folding test revealed that in vitro, HLA-A * 11: 01 and β2-microglobulin and 29 EBV-specific CTL epitope candidate peptides were combined to form MHC-monomer. In order to confirm whether these 29 types of EBV LMP2-specific or EBNA1-specific CTL epitope candidate peptides bind to HLA-A * 11: 01 and to recognize CTLs present in vivo, It is necessary to examine whether it is possible to amplify EBV-specific CTL present in peripheral blood using peripheral blood of a donor carrying HLA-A * 11: 01. First, it was confirmed by genotyping of HLA-A using Genosearch (trademark) HLA-A Ver. 2 (MBL) whether or not a donor carries HLA-A * 11: 01. Subsequent studies were performed using PBMC of 5 healthy adults carrying HLA-A * 11: 01.

[Preparation of Antigen Presenting Cells]
(1) Preparation of EBV-Infected B Cell Line Standard method (Kuzushima K, Yamamoto M, Kimura H, Ando Y, Kudo T, Tsuge I, Morishima T. Establishment of anti-Epstein-Barr virus (EBV) cellular immunity by adoptive transfer of According to virus-specific cytotoxic T lymphocytes from an HLA-matched sibling to a patient with severe chronic active EBV infection. Clin Exp Immunol. 1996; 103: 192-198), B95.8 cell line (JCRB Cell cell) which is an EBV producing cell line The culture supernatant (obtained from Bank) (containing live EBV virus) and PBMC were mixed and cultured to establish an EBV-infected B cell line (Lymphoblastoid cell line, hereinafter referred to as EBV-infected LCL). About two weeks later, the expression of HLA molecules, and the expression of CD80, CD83, CD86 and CD20 were confirmed.

(2) Preparation of CD40-B Cells NIH 3T3 cells (NIH-CD40L) into which human CD40L was introduced and stably expressed were cocultured with PBMC in the presence of IL-4. NIH-CD40L was growth-inhibited by X-ray irradiation of 96 Gy, and co-culture was repeated every 3 to 4 days (Kondo E, Topp MS, 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). About two weeks later, the expression of the HLA molecule and the expression of CD80, CD83 and CD86 were confirmed.

(3) Preparation of dendritic cells
The PBMCs were cultured in a plastic culture dish at 37 ° C. in a 5% CO ₂ thermostat for 2 hours to gently wash away cells that did not adhere to the culture dish. GM-CSF and IL-4 were added to this, followed by culturing for 24 hours, followed by adding TNFα, IL-1β and PGE2 (Prostaglandin E2), and culturing for 24-48 hours. The cells recovered by light washing with an appropriate medium etc. were made dendritic cells (Kondo E, Topp MS, 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). After 48 hours, the expression of HLA molecules, and the expression of CD80, CD83 and CD86 were confirmed.

[Induction of EBV-specific CTL]
1) Induction using antigen presenting cells
The aforementioned antigen-presenting cells (EBV-infected LCL, CD40-B cells, dendritic cells) were prepared in advance from PBMC retaining HLA-A * 11: 01. Antigen presenting cells are suspended in pulse medium (0.1% human serum albumin / 55 μM 2-mercaptoethanol / RPMI 1640) or AIM-V medium (Invitrogen), and CTL epitope candidate peptides are suspended at a concentration of 10 μg / mL. In addition, after leaving for 30 to 60 minutes at room temperature with gentle mixing at intervals of approximately 15 minutes, wash 3 times with excess washing solution (2% fetal calf serum (FCS) / PBS) and bind to no HLA molecule Was washed away. 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 cells subjected to this manipulation are called peptide-pulsed antigen-presenting cells. Peptide-pulsed antigen-presenting cells were subjected to lethal X-ray irradiation or mitomycin C treatment to lose their growth ability. This was mixed with PBMCs isolated from the same person, or CD8 positive or CD4 positive T cells, and cultured in a 37 ° C., 5% CO ₂ thermostat bath. The medium used is 10% FCS-containing RPMI 1640 medium, 10% human serum-containing RPMI 1640 medium, or 1 to 10% human plasma-containing RPMI 1640 medium, etc. In this method, 10% human serum-containing Good results were obtained with RPMI 1640 medium. Although IL-2 (Shionogi Pharmaceutical Co., Ltd.) was added to support the maintenance of T cell survival and proliferation, the timing is usually reported to be 7 to 14 days after the start of mixed culture. -2 was examined at the start of culture, 2 days after start of culture, 6 days after start of culture, but good results were obtained when administered 2 days after start of culture, so the concentration of 50 U / mL 2 days after start of culture Added. Stimulation was applied again using peptide-pulsed antigen-presenting cells 10 to 14 days after the initiation of culture. Stimulation was performed using peptide-pulsed antigen-presenting cells every 10-14 days, and evaluation of CTL induction was performed after approximately 2 weeks and 4 weeks. When the induction of EBV-specific CTL could be confirmed, peptide pulse antigen-presenting cells were further used for stimulation to establish a CTL line.

2) Induction method without using antigen-presenting cells This induction method is a method of introducing a peptide into PBMC culture solution to induce CTL. It is thought 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, it is advantageous that it can be distinguished and conveniently performed in that there is no need to prepare antigen-presenting cells in advance. It is a system that stimulates and propagates memory / effector type CTL circulating in peripheral blood by intentionally not using antigen presenting cells.

Peripheral blood collected from healthy adults holding HLA-A * 11: 01 was centrifuged at 3,000 rpm for 5 to 10 minutes, and the plasma portion of the supernatant was recovered. PBMCs were separated according to the conventional method except for the plasma part. It is characterized by adding several% of plasma to the medium used for induction. In the present invention, good results were obtained when 5% of plasma was added. The medium is generally added to the medium used for cell culture with the appropriate additives and antibiotics. As a CTL induction medium used in the present invention, a medium obtained by adding 2-mercaptoethanol, L-glutamine, and streptomycin and penicillin as antibiotics to RPMI 1640 Hepes modify (Sigma) was used. Besides this, insulin, transferrin, selenious acid, pyruvic acid, human serum albumin, non-essential amino acid solution and the like can also be added. 1 to 3 × 10 ⁶ PBMC were suspended in 1 to 2.5 mL of medium. To this was added the candidate peptide at a concentration of 1-20 μg / mL. The concentration of peptide can vary depending on the solubility of the peptide. In the present invention, it was carried out at 10 μg / mL. Two days later, addition of IL-2 was performed at a final concentration of 20-100 U / mL. For mixed culture of PBMC and peptide, it is desirable to use a carbon dioxide-exchangeable round bottom culture dish. In the present invention, a 14 mL round bottom tube made of polypropylene (BD Biosciences) or a 96 well U bottom cell culture is used Microtest plate (BD Biosciences) was used. Confirmation of EBV-specific CTL was performed after 2 weeks and 4 weeks of culture. After two weeks of culture, the cells were again stimulated with 10 μg / mL of peptide. When the induction of EBV-specific CTL could be confirmed, peptide pulse antigen-presenting cells were used for stimulation to establish a CTL line.

[Confirmation of EBV-specific CTL]
Whether or not EBV-specific CTL were present in the cell population cultured by the above-mentioned method was examined by intracellular IFNγ-producing cell assay, MHC-tetramer assay.

Confirmation of Specific CTL Induction by Intracellular IFNγ-Producing Cell Assay About 1/10 to the whole of the cell population derived by the above method is transferred to a 96-well U-bottom cell culture microtest plate, and the peptide used for induction is finalized. It was added at a concentration of 0.01 to 0.05 μg / mL. Furthermore, an intracellular protein transport inhibitor (for example, Brefeldin A, Monensin etc.) was added, and the cells were cultured in a 5% CO ₂ thermostat at 37 ° C. for 5 to 16 hours. After culture, the cells were washed, PE (phycoerythrin) -labeled MHC-tetramer reagent and PC5 (phycoerythrin-Cy5) -labeled anti-CD8 antibody (Beckman Coulter) were added, and left at room temperature for 15 to 30 minutes. After washing, it was fixed with 4% formaldehyde at 4 ° C. for 15 minutes, and then washed with an excessive amount of washing solution. After permeabilizing with 0.1% saponin, FITC-labeled anti-IFNγ antibody (Beckman Coulter) was added and reacted at room temperature for 15 to 30 minutes. After washing, the percentage of IFNγ positive cells in T cells or the percentage of IFNγ positive cells in MHC-tetramer reagent positive cells was quantified using a flow cytometer.

  FIG. 4 shows the results of using a positive control peptide to induce specific CTL by an induction method that does not use antigen-presenting cells, and examining it by an intracellular IFNγ-producing cell quantification method. Epitope peptide derived from HLA-A * 11: 01-restricted CMV pp65 (ATVQGQNLK, SEQ ID NO: :) isolated from healthy adult peripheral blood that retains HLA-A * 11: 01 but not HLA-A * 24: 02 34) or each of the peptides used for stimulation after stimulation for 13 days with an epitope peptide (AYACNTSTL, SEQ ID NO: 35) derived from HLA-A * 24: 02-restricted survivin-2B as a negative control peptide in the presence of Brefeldin A 14 I re-stimulated time. The results of triple staining with PE labeled MHC-tetramer reagent (MBL), PC5-labeled anti-CD8 antibody, and FITC-labeled anti-IFNγ antibody and analysis using a flow cytometer are shown. The numbers in the dot plot development figure show the ratio (%) of the cells present in the quadrants to the total living cells. The four divided areas are hereinafter referred to as UL (upper left), UR (upper right), LL (lower left), and LR (lower right). In the dot plot developed plot of the fluorescence intensity for CD8 on the X axis and the fluorescence intensity for IFNγ on the Y axis on the log scale, IFNγ positive CD8 positive cells appear in UR when restimulated with a positive control peptide, and reintroduced with the negative control peptide. Even if it is stimulated, it hardly appears. The presence of CMV pp65-specific CTL in a cell population stimulated and cultured with a positive control peptide for 13 days is indicated by the dot scale of the fluorescence intensity against CD8 on the X axis and the MHC-tetramer reagent on the Y axis on a log scale. The figure is clear from the presence of CD8 positive MHC-tetramer reagent positive cell population in UR. On the other hand, it is also clear that survivin-2B-specific CTLs do not appear in the cell population to which a restricted negative control peptide different from the donor allele is added. That is, in the cell population to which the epitope peptide derived from HLA-A * 11: 01-restricted CMV pp65 was added, 29.37% of specific CTL was induced, and the HLA-A * 24: 02-restricted epitope peptide derived from survivin-2B was used. Specific CTLs are not induced in the added cell population. This result is consistent with the above measurement results of IFNγ. Furthermore, in the dot plot development graph in which the fluorescence intensity for the MHC-tetramer reagent is shown on log scale on the X axis with IFNγ and Y axis, there are almost no MHC-tetramer reagent positive and IFNγ positive cells when the negative control peptide is added. Although not, specific CTL induced by stimulation of a positive control peptide derived from HLA-A * 11: 01-restricted CMV pp65 accounts for 29.39% of the total cell population, of which 73% produce IFNγ ( UR), in other words, having cytotoxic activity was revealed.

  From this result, in PBMCs cultured with the addition of HLA-restricted CTL epitope peptide retained by the donor, restimulation induces IFNγ-producing cells, which are stained with the MHC-tetramer reagent. It is clear that it is a CTL specific for the added CTL epitope peptide. It is also possible to determine whether or not such a specific CTL is induced by the intracellular IFNγ-producing cell assay. Similarly, an investigation of whether IFNγ-producing cells were induced using EBV LMP2-specific CTL epitope candidate peptides was performed in 5 healthy adults. The percentage of IFNγ-producing viable cells present in CD8 positive cells was quantified and shown in FIG. FIG. 6 shows representative results of quantification of intracellular IFNγ-producing cells.

  In FIG. 5, the peripheral blood of five healthy adults (donor ID numbers: * 11-13, * 11-16, * 11-8, * 11-11, * 11-2) is used and 21 types of peptides are used. , Intracellular IFNγ producing cells were quantified. The percentage (%) of the number of CD8-positive IFNγ-positive cells in PBMC is shown numerically on the X-axis. The peptide candidates that showed a positive as compared to the negative control are ASS (10 mer) (SEQ ID NO: 1) and the positive control peptide CMV pp65. The detailed result of the peptide which showed positive is demonstrated in FIG.

  In FIG. 6, the dot plot developed with CD8 on the X axis and the fluorescence intensity for IFNγ on the Y axis in log scale, and restimulation with the same peptide as the peptide used for induction to quantify intracellular IFNγ producing cells Showed the results. In UR, the percentage (%) of the number of CD8 positive IFNγ positive cells in PBMC was shown numerically.

  In CMV pp65 as a positive control, 28.0% of CD8 positive IFNγ positive (producing) cells were detected in UR. A significant difference was observed compared to the positive rate (8.67%) in the case of the negative control survivin-2B. Based on these results, MHC-tetramer reagents were synthesized, and their usefulness was examined.

[Study using EBV-specific MHC-tetramer reagent]
(Synthesis of MHC-tetramer reagent)
We made PE-labeled MHC-tetramer reagents using EBV-specific CTL epitope peptides and HLA-A * 11: 01 molecules. The MHC-tetramer reagent prepared in the present invention is, for example, abbreviated as ASS (10 mer) -Tet, which comprises HLA-A * 11: 01 molecule and peptide ASS (10 mer) (ASSYAAAQRK, SEQ ID NO: 1) The thing synthesize | combined using the ternary complex of (beta) 2-micro globulin is shown.

[Quantification method using MHC-tetramer reagent]
Using an MHC-tetramer reagent prepared using the CTL epitope peptide of the present invention, the CTL line obtained by adding the CTL epitope peptide to PBMCs isolated from peripheral blood or peripheral blood is EBV-specific as a sample Quantitative determination of CTL. An example is shown below for the case where PE labeled MHC-tetramer reagent is used. The labeling dyes may be used in appropriate combinations according to the type of flow cytometer to be used, and are not limited to the following.

In the case of peripheral blood: To 200 μL of peripheral blood collected, 10 μL of PE-labeled MHC-tetramer reagent, 20 μL of FITC-labeled anti-T cell surface antibody (eg, CD8, CD4, CD3) and the like were added. Furthermore, in order to exclude nonspecific fluorescence due to contaminating red blood cells, an anti-CD45 antibody labeled with PC5 or the like may be added. Gently mixed and left at room temperature for 30 minutes. OptiLyse B (Beckam Coulter) was added and hemolytic fixation was performed according to the standard. After 2 mL of PBS was added and stirred, it was centrifuged at 400 × g for 5 minutes. After the supernatant was aspirated and discarded, the cells were resuspended in 500 μL of PBS and analyzed by flow cytometer within 24 hours.

In the case of CTL lines derived using PBMC or CTL epitope peptide 10 μL of PE-labeled MHC against an appropriate amount of PBMC (10 ⁵ to 10 ⁶ ) or an appropriate amount of CTL line derived using a CTL epitope peptide -A tetramer reagent, 20 μL of FITC-labeled anti-T cell surface antibody (eg, CD8, CD4, CD3) and the like were added. Furthermore, in order to exclude nonspecific fluorescence due to contaminating red blood cells, an anti-CD45 antibody labeled with PC5 or the like may be added. Gently mixed and left at room temperature for 30 minutes. After 3 mL PBS was added and stirred, it was centrifuged at 400 × g for 5 minutes. After aspiration and discarding the supernatant, cells were resuspended in 500 μL PBS. In the case of a CTL line, 7-AAD viability Dye (dead cell detection reagent, MBL) may be added to exclude nonspecific fluorescence by dead cells. Analysis was made with a flow cytometer within 24 hours.

[Detection of EBV LMP2 specific CTL in healthy adult peripheral blood by MHC-tetramer reagent]
A person whose immunity has been reduced due to any cause, a patient with congenital immunodeficiency, or a patient who has been receiving an immunosuppressant for bone marrow transplantation, hematopoietic stem cell transplantation, cord blood transplantation, solid organ transplantation and rejection prevention To know whether EBV-specific CTL is present in the peripheral blood of patients with chronic viral infections, patients with AIDS, patients with high risk such as elderly people, children and pregnant women, or transplant donors, it is an antiviral agent Important in the management of infections including the proper use of drugs and immunosuppressants. However, since the immunocompetence of these patients is reduced, the presence of EBV-specific CTLs is also reduced, and it was difficult for the existing methods to be present. If EBV-specific MHC-tetramer reagent is used, it may be possible to determine the presence of EBV-specific CTL in about one hour after blood collection. Therefore, we examined whether healthy adult peripheral blood could be used to detect EBV-specific CTL. The results are shown in FIG. Three EBV-specific MHC-tetramer reagents and positive controls CMV pp65 MHC-tetramer reagent (MBL) and negative controls survivin-2B HLA-A * 24: 02 using donor ID * 11-11 peripheral blood The cells were stained with MHC-tetramer reagent (MBL). The fluorescence intensity with respect to the MHC-tetramer reagent on CD8 on the X axis and Y axis is shown in a dot plot development diagram analyzed on a log scale. The numerical values in the dot plot development show the percentage of CD8 positive MHC-tetramer reagent positive cells in CD8 positive cells as a positive rate (%). As a result, the cell population positive for CD8 positive MHC-tetramer reagent immediately after blood collection (day 0) was not significantly different from the negative control. This is because the peripheral blood used is a healthy adult and it is less likely to develop an opportunistic infection with EBV or CMV. Subsequently, PBMC were incubated with the respective peptides for 14 days (day 14) and then stained similarly with the MHC-tetramer reagent. As a result, in the culture for 14 days, positive ratios of 44.47% for ASS (10 mer) and 40.52% for CMV pp65 were detected. The cell population that showed positive even when the positive rate obtained with the negative control HLA-A * 24: 02 restricted survivin-2B MHC-tetramer reagent was 0.17% is a CTL population specific for each peptide It can be said that From these results, it was revealed that EBV LMP2-specific CTLs are present in peripheral blood of healthy adults and can be proliferated to such an extent that the presence or absence of CTLs can be determined in two weeks of culture. This means that the induction method carried out in the present invention which does not utilize antigen-presenting cells is effective for detection of EBV-specific CTL. In addition, EBV LMP2-derived ASS (10 mer) identified in the present invention (SEQ ID NO: 1) has a function of proliferating EBV LMP 2 specific CTL in peripheral blood, and these cell populations are ASS (10 mer) -Tet. It was found that it was an HLA-A * 11: 01-restricted EBV LMP2 specific CTL epitope peptide because it was detectable by

[Detection of EBV EBNA1 specific CTL in healthy adult peripheral blood by MHC-tetramer reagent]
Subsequently, we performed the identification of EBV EBNA1-derived HLA-A * 11: 01 restricted CTL epitopes. From the verification results obtained in the previous LMP2, as shown in FIG. 6, 8.67% non-specific reaction was detected in the negative control survivin-2B as shown in FIG. On the other hand, as shown in FIG. 7, in the detection of CTL using an MHC-tetramer reagent, it became clear that there is almost no nonspecific reaction.

  Since no reports of CTL epitopes derived from HLA-A * 11: 01-restricted EBV EBNA1 have existed in the past, it was decided to carry out verification by MHC-tetramer staining method with less nonspecific staining.

  As shown in Table 5, as a result of the folding test, all 11 EBV EBNA1 specific CTL epitope candidate peptides showed HLA * 11: 01 binding. Therefore, in order to identify EBV EBNA1 specific CTL epitope peptides, the inventors produced MHC-tetramer reagents using eleven EBV EBNA1 CTL epitope candidate peptides. Subsequently, it was examined whether EBNA1-specific CTL could be detected using healthy adult peripheral blood. The results are shown in FIG. 8 and FIG. First, peripheral blood of five healthy adults (donor ID numbers: * 11-13, * 11-16, * 11-8, * 11-11, * 11-2) using a mixture of three types of peptides. CTL induction was performed using Two weeks later, detection was carried out using three mixed MHC-tetramer reagents corresponding to the inducing peptide. In FIG. 8, the dot plot developed view in which the fluorescence intensity for the tetramer is analyzed on the log scale with CD8 on the X axis and the Y axis is shown. The numerical values in the dot plot development view represent the proportion of MHC-tetramer reagent positive cells in CD8 positive cells in living cells as a positive rate (%). As a result, in the donor * 11-8, a mixture of three MHC-tetramer reagents (HRG) synthesized using the same three kinds of peptides and a cell population derived using the (HRG + GVF + KTS) ternary mixed peptide When stained with -Tet + GVF-Tet + KTS-Tet), 0.38% positive cells were detected. In order to verify which of the three peptides there is specificity for, in FIG. 9, detection was separately performed using each of the three MHC-tetramer reagents. As a result, when stained with HRG-Tet alone, HRG-Tet positive cells were detected at a positive rate of 0.42%. Since no positive cells were detected in Tet-GVF-Tet and KTS-Tet, HRG (SEQ ID NO: 26) is an EBV EBNA1-specific CTL epitope peptide that exhibits HLA-A * 11: 01 restriction It has been shown.

[Confirmation of EBV EBNA1-specific CTL induction by intracellular IFNγ-producing cell assay]
The production of intracellular IFNγ is used as one indicator of the cytotoxic activity of CTL. In order to evaluate the functionality of CTL induced by EBV EBNA1 specific CTL epitope peptide identified by the above-mentioned MHC-tetramer reagent, the inventors performed intracellular IFNγ-producing cell quantification. The results are shown in FIG.

  The upper part of FIG. 10 is a dot plot development view showing the CD8 on the X axis and the fluorescence intensity for IFNγ on the Y axis in log scale, and restimulation with the same peptide as the peptide used for induction to produce IFNγ production ability The results of the verification are shown. In UR, the percentage (%) of the number of IFNγ positive cells in CD8 positive cells in PBMC was shown numerically. The lower part of FIG. 10 shows the result of staining with a tetramer when the IFNγ production ability was verified. In a dot plot developed plot of the fluorescence intensity for tetramer on a log scale with CD8 in the X axis and the Y axis in the X axis, the percentage (%) of the number of tetramer positive cells in CD8 positive cells in PBMC is shown in UR.

  HRG-induced CTLs were restimulated for 14 hours with HRG peptide or survivin-2B peptide as a negative control. As a result, CD8 positive IFNγ positive (producer) cells (restimulated: 0.08%; unstimulated: 0.06%) barely appeared when restimulated with the negative control survivin-2B and when not stimulated. Restimulation with the HRG (SEQ ID NO: 26) peptide resulted in the appearance of 0.68% CD8 positive IFNγ positive (producer) cells in UR (0.98% CD8 positive tetramer positive cells).

  From the above, EBV EBNA1-derived HRG (SEQ ID NO: 26) identified in the present invention has the function of proliferating EBV EBNA1-specific CTL in peripheral blood, and these cell populations have cytotoxic activity and It was found that HRG-Tet was detectable as HLA-A * 11: 01-restricted EBV EBNA1 specific CTL epitope peptide.

[Study on importance of amino acids at N-terminal and C-terminal of ASS (10 mer) sequence]
The CTL epitope peptide presented to the HLA class I molecule consists of 8 to 10 amino acids, and the second from the N-terminal side, and the 9th or 10th amino acids are most important for binding to the HLA class I molecule It is an amino acid and is called an anchor motif. As a peptide that binds to the HLA-A11 molecule, any of Ile, Met, Ser, Thr, or Val is placed at the second position from the N-terminus, and either Lys or Arg is located at the ninth or tenth position. The arranged peptide consisting of 9 to 10 amino acids is the best known (Chang CX, Tan AT, Or MY, Toh KY, Lim PY, Chia AS, Froesig TM, Nadua KD, Oh Eur J Immunol. 2013 Apr; 43. HL, Leong HN, Hadrup SR, Gehring AJ, Tan YJ, Bertolletti A, Grotenbreg GM. (4): 1109-20.). The HLA-A * 11: 01-restricted EBV LMP2 specific CTL epitope peptide ASS (10 mer) provided by the present invention is composed of 10 amino acids: Ala Ser Ser Tyr Ala Ala Ala Gln Arg Lys (SEQ ID NO: 1) It is composed and has Ser from the N-terminal side and Ser from the N-terminal side as an anchor motif, Arg at the 9th and Lys at the 10th. Therefore, in order to clarify the embodiment of the present invention, a peptide in which one amino acid residue was deleted from the N-terminal side or from the C-terminal side was synthesized and verified. The amino acid sequence of SEQ ID NO: 6 from which Ala at the N-terminal side is deleted is Ser Ser Tyr Ala Ala Ala Gln Arg Lys, and the amino acid sequence of SEQ ID NO: 5 from which Lys at the C-terminal side is deleted is Ala Ser Ser Tyr Ala Ala Ala Gln Arg. The results of comparative experiments using these are shown in FIGS.

Ala Ser Ser Tyr Ala Ala Ala Gln Arg Lys ASS (10 mer) SEQ ID NO: 1
Ala Ser Ser Tyr Ala Ala Ala Gln Arg ASS (9 mer) SEQ ID NO: 5
Ser Ser Tyr Ala Ala Ala Gln Arg Lys SSY SEQ ID NO: 6

  In FIG. 11, a folding test was performed to examine the ability to form a complex with HLA-A * 11: 01. As a result, according to the result of the folding test for 7 days, the MHC-monomer formation rate is ASS (9 mer) (337, 803 μV * sec) in descending order, SSY (294, 932 μV * sec), ASS (10 mer) The ability to form an MHC-monomer was observed in comparison with the peptide of negative control (83, 587 μV * sec).

  In the experiment of FIG. 12, CTL induction experiments were carried out using three types of peptides in the above-described induction method not using antigen-presenting cells. Detection of each specific CTL was carried out using 3 kinds of MHC-tetramer reagents 14 days after induction. The upper part of FIG. 12 induced CTL using ASS (10 mer), which was stained using ASS (10 mer) -Tet, ASS (9 mer) -Tet and SSY-Tet. As a result, when stained with ASS (10 mer) -Tet, 53.31% of ASS (10 mer) -Tet positive CD8 positive specific CTLs were detected, but ASS (9 mer) -Tet or SSY-Tet positive CD8 positive No specific CTL was detected (upper middle and upper left). The upper cell population can not be detected by ASS (9 mer) -Tet or SSY-Tet, despite the presence of 53.31% of ASS (10 mer) -Tet positive CD8 positive cell populations. It turned out that the ASS (10 mer) -Tet positive CD8 positive cell population can not recognize the ASS (9 mer) and SSY presented on the HLA-A * 11: 01 molecule. Similar experiments were performed on CTL-induced cell populations with ASS (9 mer) and SSY peptides, but any cell population is reactive to ASS (10 mer) -Tet, ASS (9 mer) -Tet, SSY-Tet No cell population was detected (middle and lower). This indicates that, as shown in FIG. 11, although both ASS (9 mer) and SSY peptides have the ability to bind to HLA-A * 11: 01 molecules, the EBV LMP2-specific CTL precursor present in blood ( ASS (10 mer) means CTL induction even if any one of N-terminal Ala residue or C-terminal Lys residue is deleted. It became clear that the ability and CTL detection ability were lost.

  In FIG. 13, the ability to produce IFNγ when restimulated with the same peptide as the peptide used for induction was detected in the CTL-induced cell population using each of the ASS (10 mer), ASS (9 mer), and SSY peptides. Showed the results. In the cell population derived 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, there are 40.52% of ATV-Tet positive cells, 28% of cells produced IFNγ upon restimulation with ATV peptide.

  On the other hand, in the case of the HLA-A * 24: 02 restricted epitope peptide (AYACNTSTL, SEQ ID NO: 35) of survivin-2B used as a peptide for negative control, 0.17% of AYA-Tet positive cells were detected. The cell population is not detected as a clear spot and is considered as a nonspecific reaction. In addition, 8.67% of IFNγ positive cells were detected by restimulation with AYA peptide, which is a level of nonspecific staining common in intracellular IFNγ staining. In the cell population derived using EBV LMP 2-derived HLA-A * 11: 01-restricted ASS (10 mer) identified in the present invention, 53.31% of ASS (10 mer) -Tet specific CTL exist, and ASS (10 mer) 68% of the cell population produced IFNγ by restimulation of On the other hand, in the case of SSY in which the N-terminal Ala residue was deleted, SSY-Tet positive cells were not detected, and IFNγ production was also lower than that of the negative control. From this, it is a peptide having an amino acid corresponding to the anchor motif, and even in the case of forming an MHC-monomer, it is not always possible to specifically induce CTL as an epitope peptide, yet it is specific It has been shown that an epitope peptide capable of inducing CTL in can not be easily conceived.

[From mutation of amino acids of CTL epitope to EBV strain diversity to peptide cocktail therapy]
(Amino acid mutation of CTL epitope of ASS (10 mer) between EBV strains)
Amino acid mutations of CTL epitopes due to the diversity of EBV strains cause a reduction in immune response and escape, which is a potential problem of immunotherapy using CTL epitopes. In order to solve this problem, peptide cocktail therapy in which a plurality of CTL epitopes are mixed is being proposed. This can be said to be a solution that is expected to be able to be compensated by another epitope if it is an immune non-response due to mutation or the like when one type of epitope is used. As this solution, a method of using a plurality of CTL epitopes for one kind of HLA type and a method of using each CTL epitope for a plurality of HLA types can be considered, but desirably, by combining the both. is there. For example, if the subject patient retains HLA-A2 and HLA-A11, it is desirable that multiple HLA-restricted CTL epitopes be selected for treatment. This is one of the problems to be solved by the present invention.

  As mentioned earlier, it has become clear in previous studies of several research groups that NPC patients have the highest number of HLA-A11 carriers. However, so far, only one SSC has been reported for HLA-A11 restricted CTL epitopes of LMP2 and EBNA1.

  The inventors examined amino acid mutations between strains for ASS (10 mer) which is an HLA-A * 11: 01 restricted LMP2-derived CTL epitope and HRG which is an HLA-A * 11: 01 restricted EBNA1-derived CTL epitope The results are shown in FIG. Novel epitopes ASS (10 mer) are GD2 (from Guangdong, China) and HKNPC1 (from Hong Kong NPC patients), and the second Ser residue and the fourth Tyr residue are mutated to Asn and Ser residues, respectively It turned out that (S2N Y4S) was done. Furthermore, the novel epitope HRG was found to be mutated in two strains. In AG 876 (West African type 2), the fourth Gln residue is mutated to a Glu residue, the eighth Asn residue to a Ser residue, and the ninth Pro residue to a Gln residue. In Mutu (from Asia), the 9th Pro residue is mutated to the Gln residue.

[Sequence of ASS (10 mer) CTL epitope in each sample]
In order to investigate the mutation of the CTL epitope of ASS (10 mer) newly identified in the present invention in detail, the inventors used five donors (* 11-2, * 11-8, * 11-11) used in the present invention. , * 11-13, * 11-16), analysis of EBV LMP2-derived DNA sequences including ASS (10 mer) was performed. The primers for DNA sequence analysis are designed and amplified in Exon 2 of LMP2 and the gene product to be amplified is 292 bp.
5'primer: TTGCTCTATTCACCCTTACT (SEQ ID NO: 50)
3'primer: ATGCATTGTAAATGGTGCGT (SEQ ID NO: 51)

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, the Exon 2 fragment was amplified by PCR reaction using the above-mentioned primers, and cloned using TOPO (registered trademark) TA cloning Kit (Life technologies). Three clones were selected from each sample for sequence analysis. The results of the sequence are shown in FIG. In FIG. 15, the mutation position of ASSYAAAQRK (SEQ ID NO: 1) (base sequence: GCCAGCTCATATGCGCGCTGCACAAAGGAA (SEQ ID NO: 48), GCCAGCTCATATGCGCGCTGCACAGAGGAAA (SEQ ID NO: 49)) observed between EBV strains is shown by close-up. Table 6 is a sequence result of ASS (10 mer). 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, the five Japanese donors used in the present invention were infected with the most widely distributed B95.8 strain, and no mutation of the ASS (10 mer) CTL epitope could be detected. Based on the result of the above sequence, the inventors examined the influence of mutation of ASS (10 mer) CTL epitope found among EBV strains on CTL inducibility. Synthesized peptides ASS (10 mer) (SEQ ID NO: 1), ANS (S2 N) (SEQ ID NO: 2), ASS (Y 4 S) (SEQ ID NO: 3), ANS (S 2 N; Y 4 S) (SEQ ID NO: 4) CTL induction was performed using donor * 11-11 PBMCs using survivin-2B as a control peptide. The results are shown in FIG. In the induction using ASS (10 mer), 27.63% of ASS (10 mer) -Tet positive cells were detected, but in each mutant peptide, no induction of CTL was observed. This is considered to be because donor * 11-11 is an infected person of strain B 95.8 according to sequence analysis of EBV LMP 2 and no mutation of amino acid is found in ASS (10 mer). It is also specific to ANS (S2N) (SEQ ID NO: 2), ASS (Y4S) (SEQ ID NO: 3) and GD2 and HKNPC1 strain ANS (S2N; Y4S) against B95. The failure to induce CTL revealed that the S2N and Y4S mutations are important mutations for TCR recognition. Therefore, when inducing EBV-specific CTL, it is important to examine whether there is a mutation in the region of ASS (10 mer) in advance, and if there is a mutation, ANS (S2N), ASS (Y4S), ANS It is considered preferable to use (S2N; Y4S).

[Difference between CTL inducibility and TCR binding ability by mutation of ASS (10 mer) CTL epitope]
In order to investigate in detail the influence on TCR binding ability by mutation of S2N and Y4S, we used ANS (S2N) -Tet, ASS (Y4S) -Tet, ANS (S2N) using three kinds of mutant sequence peptides. Three different MHC tetramer reagents, Y4S) -Tet, were prepared. Tetramer staining for PBMC-derived HLA-A * 11: 01-restricted EBV LMP2-specific CTL was performed using the ASS (10 mer) epitope peptide (SEQ ID NO: 1) provided in the present invention. The results are shown in FIG.

  As HLA-A * 11: 01-restricted EBV LMP2 ASS (10 mer) specific CTL was detected by ANS (S2N) -Tet as shown in FIG. 17, ANS (S2N) -Tet was ASS (10 mer). )-Although cross reactivity with Tet was maintained (26.87% vs. 27.63%), almost no positive cells could be detected in ASS (Y4S)-Tet and almost no binding to TCR ( 0.49%). With ASN (S2N, Y4S) -Tet, the ability to bind to TCR was slightly retained (5.93%). This is considered to be due to the difference in the degree of change in hydrophobicity / hydrophilicity of amino acids and the difference in the configuration of side chains due to both substitution of S2N and Y4S and substitution of Y4S alone. For the two amino acid mutations, there was no single substitution between EBV strains and only co-substitution was found (GD2, HKNPC1). However, it should be noted that the mutations of ASS (10 mer) provided in the present invention can be found only in the GD2 strain and the HKNPC1 strain. From this, it is considered that this epitope is functionally complementary, that is, it is possible to avoid the reduction of the immune response by the amino acid mutation caused by the difference between the GD2 strain and the HKNPC1 strain and the other infected EBV strains by using both of them.

  Furthermore, with regard to the novel epitope HRG (SEQ ID NO: 26) derived from HLA-A * 11: 01-restricted EBV EBNA1, HRG derived from AG876 strain (Q4E, N8S, P9Q) and HRG derived from Mutu strain (P9Q) are also included. Thus, as shown in FIG. 3, there was binding to HLA-A * 11: 01, and each MHC-tetramer reagent was successfully synthesized. With the same verification as ASS (10 mer), it seems that the difference in induction efficiency becomes clear for each infected strain.

[Mass culture of CTL using culture bag]
In the present invention, EBV LMP2-specific or EBV EBNA1-specific novel CTL epitopes are successfully identified, enabling the realization of antigen-specific cytotoxic T cell therapy (CTL therapy) for HLA-A11 carriers. The CTL therapy is expected as a next-generation immunotherapy. However, because preparation of antigen-specific CTLs in vitro involves complicated and complicated operations, it has been necessary to rely on an open culture system. Therefore, a medical grade culture cell processing center (CPC) had to maintain and manage a facility for maintaining a class 100 clean environment, which required a large cost.

  Moreover, since various methods are used in the conventional method, the cost required for preparation is also considerable. Furthermore, although prepared in a facility maintained at the class 100 level, culture by the open system involves a safety risk. As described above, in the conventional antigen-specific CTL preparation method, operability, economy and safety greatly hinder the practical use, and it could be implemented only in limited facilities. In order to solve the above-mentioned problems, the inventors have studied a large-scale culture method of CTL, and examined an EBV-specific CTL culture system which is simple and safe and which can be implemented at low cost. Large-scale culture of EBV LMP2-specific CTL was successfully performed in as little as three weeks using a commercially available gas-permeable culture bag.

  FIG. 18 shows the results of microscopic observation of cells during the culture period. Anti-CD3 antibody-stimulated PBMCs were observed on day 7 and peptide-stimulated PBMCs on day 14. Activated T cell clusters were observed, and one week after starting mixed culture (day 21), activated T cell clusters were observed. It was shown that they further proliferated.

  As mentioned above, the use of mixed peptides is one of the solutions to enhance the effectiveness of cancer immunotherapy. In this example, four kinds of peptides of the newly identified CTL epitope ASS (10 mer) (SEQ ID NO: 1) and its mutant sequence ASN (S2N) (SEQ ID NO: 2) were mixed, and CTL large scale culture was performed. The results are shown in FIGS.

  FIG. 19 shows the total cells and the number of CTLs proliferated with four peptide induction during the culture period. The number of CTLs was calculated by the tetramer staining results of FIG. FIG. 19 is a dot plot development view in which the fluorescence intensity for tetramer is shown in log scale on CD 8 on the X axis and on the Y axis, and stained with the tetramer of the same peptide as the peptide used for induction. The numerical values in the dot plot development view represent the proportion of MHC-tetramer reagent positive cells in CD8 positive cells in living cells as a positive rate (%).

For ASS (10 mer) and ANS (S2N), the number of MHC-tetramer reagent positive cells in isolated PBMCs was only 0.04% or less before mass culture, but the day 7 to day 24 when positive cells were detected Among them, ASS (10 mer) was confirmed to have about 100-fold growth from 1.3 × 10 ³ to 2.3 × 10 ⁵ and ANS from 3.2 × 10 ³ to 3.1 × 10 ⁵ . This demonstrates the efficacy of the peptide cocktail therapy with a novel CTL epitope peptide. Furthermore, the success of mass proliferation of CTLs using a novel CTL epitope peptide is one that shows its practicality in clinical practice.

  The above culture system examined by the inventors is characterized in that it is performed in a closed system, not in a conventional open system. Therefore, it is considered that the operability, economy and safety in preparation of antigen-specific CTL can be realized, and the practical application of CTL therapy can be promoted.

  In the past clinical trials of cancer immunotherapy, one epitope was often used for one HLA type, but although effectiveness has been recognized, individual differences can not be ruled out. As mentioned above, cocktail peptide therapy is one of the solutions. However, only one EBV LMP2 antigen-specific and EBNA1 antigen-specific HLA-A11 restricted epitope peptide has been identified so far. It should be pointed out that the decrease in immune response that can be caused by amino acid mutations among EBV strains is a potential problem in the clinic. It was considered that this problem could not be overlooked, and as a result of intensive studies in the present invention, new epitopes ASS (10 mer) (SEQ ID NO: 1) and HRG (SEQ ID NO: 26) were identified. This novel CTL epitope is considered to be a major contribution to the treatment of EBV-related cancer and immunodeficiency, particularly NPC with many HLA-A11 patients.

[Vaccine injection]
The peptide of SEQ ID NO: 1 was each dissolved in DMSO to a final concentration of 20 mg / ml and filter-sterilized. The resulting peptide-containing solution was dispensed by 1 mL each into a sterile vial and sealed up to give a vaccine injection.

Example 2
[Prediction of CKAP4-specific HLA-A * 24: 02 restricted epitope peptide]
CKAP4 is a type II membrane protein consisting of 602 amino acids in full length, and there is no report of isoform. Selection of CKAP4 specific CTL epitope candidate peptides of the present invention was carried out against HLA-A * 24: 02 which approximately 60% of Japanese people carry. Specifically, it is possible to search for CTL epitope candidate peptides consisting of 8 to 12 amino acids having a binding motif for HLA-A * 24: 02, in comparison with a plurality of software published on the Internet Carried out. As a result, ten kinds of 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 peptides were synthesized. The following shows CTL epitope candidate peptides synthesized.

Synthesized HLA-A * 24: 02 binding CKAP4 specific CTL epitope candidate peptide
Arg Leu Gly Arg Ala Leu Asn Phe Leu Phe (SEQ ID NO: 36)
Phe Tyr Leu Ala Leu Val Ala Ala Ala Ala (SEQ ID NO: 37)
Tyr Leu Ala Leu Val Ala Ala Ala Ala Phe (SEQ ID NO: 38)
Asp Phe Ser Arg Gln Arg Glu Glu Leu (SEQ ID NO: 39)
Lys Val Gln Ser Leu Gln Ala Thr Phe (SEQ ID NO: 40)
Ser Leu Gln Ala Thr Phe Gly Thr Phe (SEQ ID NO: 41)
Thr Phe Gly Thr Phe Glu Ser Ile Leu (SEQ ID NO: 42)
Ile Tyr Thr Glu Val Arg Arg Glu Leu Val (SEQ ID NO: 43)
Lys Val Gln Glu Gln Val His Thr Leu Leu (SEQ ID NO: 44)
Asp Phe Leu Asp Arg Leu Ser Ser Leu (SEQ ID NO: 45)

  Table 7 shows the characteristics of the HLA-A * 24: 02-binding CKAP4 specific CTL epitope candidate peptides synthesized. The three or four amino acid sequences from the N-terminal side of the synthesized CKAP4 specific CTL epitope candidate peptide are shown as abbreviations as peptide names. From left, peptide name, amino acid sequence, position on CKAP4 amino acid sequence, number of amino acids, HLA peptide 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 for predicting the affinity between HLA-A * 24: 02 and the peptide, and the higher the score, it means that the HLA molecule and the peptide may form a stable complex. The inventors selected 10 types (SEQ ID NO: 36 to 45) of CTL epitope candidate peptides using analysis software such as SYFPEITHI, Rankpep, IEDB Bind prediction, NetCTL, etc. in addition to the analysis software BIMAS described in Table 7.

[Folding test of CKAP4 specific CTL epitope candidate peptide]
The inventor performed a folding test using ten kinds of CKAP4-specific CTL epitope candidate peptides artificially synthesized. Specifically, HLA-A * 24: 02 and β2-microglobulin expressed and purified using E. coli expression system, and CKAP4 specific CTL epitope candidate peptide are added to the folding solution, mixed, and then folded over time The solution was separated and analyzed on a gel filtration column. In gel filtration column analysis, when the formation of a ternary complex (MHC-monomer) of HLA-A * 24: 02 and β2-microglobulin and a CKAP4 specific CTL epitope candidate peptide is observed, the MHC-monomer is from the raw material Because of the large molecular weight, elution time in gel filtration column analysis is faster. Also, 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 in the candidate peptide which does not bind to an HLA molecule. A representative gel filtration column analysis example in which MHC-monomer formation is observed is shown in FIG.

  Although HLA molecules and β2-microglobulin are purified as inclusion bodies, which are insoluble fractions, when they are expressed and purified using E. coli expression system, they are solubilized with 8 M urea, but as a result of gel filtration column analysis The poorly soluble HLA molecules that do not lead to MHC-monomer formation are detected in 7-8 minutes as aggregates. However, most of the aggregates are removed by filter filtration before gel filtration column analysis. β 2 -microglobulin is a soluble protein, which is solubilized in the folding solution and detected around 14 minutes. After 15 minutes, the composition or peptide of the folding solution is detected. Although the peak of MHC-monomer is not confirmed immediately after the start of the folding test (day 0), the peak becomes large after 1 day (day 1) and 3 days (day 3), and MHC-monomer formation is progressing smoothly It shows the thing.

  FIG. 22 shows the analysis results after 4 days of the folding test performed on 10 types of CKAP4 specific CTL epitope candidate peptides. At the same time, a positive control peptide (SEQ ID NO: 46) consisting of 9 amino acids and a negative control (SEQ ID NO: 47) were used for comparison.

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 showed a peak area indicating MHC-monomer formation as a bar graph. As a result of comparing SEQ ID NO: 36-45 with a positive control peptide and a negative control (79, 031 μV * sec), SEQ ID NO: 36, 40, 42, 43 show good formation of MHC-monomer, and the subsequent analysis was advanced .

[Production of CKAP 4 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 NO: 36, 40, 42, 43 and HLA-A * 24: 02 and β2-microglobulin The reagent was manufactured. The MHC-tetramer reagent prepared in the present invention is, for example, abbreviated as KVQE-Tet, which comprises HLA-A * 24: 02 and peptide KVQE (KVQEQVHTLL (SEQ ID NO: 44)) and 3 of β2-microglobulin. Figure 1 shows the one produced using the human complex. An MHC-monomer, which is a complex of HLA class I molecules purified from a recombinant host for protein expression, β2-microglobulin and a CKAP4 specific CTL epitope candidate peptide of the present invention, is formed in a suitable folding solution. A biotin binding site is previously added to the C terminus of the recombinant HLA class I molecule, and biotin is added to this site after MHC-monomer formation. The MHC-tetramer reagent can be prepared by mixing commercially available dye-labeled streptavidin and biotinylated MHC-monomer at a molar ratio of 1: 4.

[Identification of CKAP4 specific CTL epitope peptide]
(Selection of HLA type of sample)
Ten candidate peptides having a binding motif for HLA-A * 24: 02 were selected as CKAP4 specific CTL epitope candidate peptides. Furthermore, in a folding test, HLA-A * 24: 02 and β2-microglobulin and four CKAP4 specific CTL epitope candidate peptides (SEQ ID NOS: 36, 40, 42, 43) bind in vitro to form MHC-monomers. It turned out that it forms well. In order to confirm whether these four CKAP4-specific CTL epitope candidate peptides bind to HLA-A * 24: 02 and there is a CTL that recognizes this in vivo, HLA-A * 24 can be used. It is desirable to verify using the peripheral blood of the donor who owns: 02. First, it was confirmed by genotyping of HLA-A using Genosearch (trademark) HLA-A Ver. 2 (MBL) whether or not the donor carries HLA-A * 24: 02. Subsequent studies were performed using PBMC of 7 healthy adults carrying HLA-A * 24: 02.

[Induction of CKAP4 specific CTL]
[Induction of CKAP4 specific CTL using MLPC (Mixed Lymphocyte-Peptide Cultures) method]
The MLPC method is a method of inducing CTL by adding a peptide to PBMC culture solution (Karanikas V, Lurquin C, Colau D, van Baren N, De Smet C, Lethe B, Connerotte T, Corbiere V, Demoitie MA) J. Immunol. 2003; 171 (9): 4898-, 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. 904, Tsukahara T, Kawaguchi S, Torigoe T, Takahashi A, Murase M, Kano M, Wada T, Kaya M, Nagoya S, Yamashita T, Sato N. HLA-A * 0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor. J Transl Med. 2009; 7: 44). It is thought that the peptide is presented to antigen-presenting cells present in PBMC, such as dendritic cells, B cells, macrophages, and certain T cells, and memory-type CTL contained in PBMC are stimulated to proliferate. Peripheral blood collected from healthy adults carrying HLA-A * 24: 02 was centrifuged at 3,000 rpm for 5 to 10 minutes, and the plasma portion of the supernatant was recovered. PBMCs were separated by conventional density gradient centrifugation except for the plasma portion. In the present invention, good results were obtained using a medium supplemented with 5% plasma. The medium is generally added to the medium used for cell culture with the appropriate additives and antibiotics. As a CTL induction medium used in the present invention, a medium obtained by adding 2-mercaptoethanol, L-glutamine, and streptomycin and penicillin as antibiotics to RPMI 1640 Hepes modify (Sigam) was used. Besides this, insulin, transferrin, selenious acid, pyruvic acid, human serum albumin, non-essential amino acid solution and the like can also be added. Approximately 3 × 10 ⁷ PBMCs were suspended in 10 mL of medium. One or two peptides out of four CKAP4 specific CTL epitope candidate peptides (SEQ ID NOs: 36, 40, 42, 43) were added to each at a concentration of 10 μg / mL. The concentration of peptide can vary depending on the solubility of the peptide. In the present invention, it was carried out at 10 μg / mL. For mixed culture of PBMC and peptide, a 96-well U-bottom cell culture microtest plate (BECTON DICKINSON) was used. The cells were cultured in a 5% CO ₂ thermostat at 37 ° C. Two days later, addition of IL-2 was performed 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 around 2 weeks of culture. When induction of CKAP4 specific CTL could be confirmed, stimulation with peptide-pulsed antigen-presenting cells or stimulation with a direct peptide was attempted to establish a CTL line.

[Confirmation of CKAP4 Specific CTL]
Examination of the presence or absence of CKAP4 specific CTL in the cell population cultured by the above-mentioned method was performed by the MHC-tetramer method. After culture, 10 μL of PE-labeled MHC-tetramer reagent, 20 μL of FITC (fluorescein isothiocyanate) -labeled T cell surface antibody (eg, CD8, CD4, CD3) and the like were added to an appropriate number of cells. Furthermore, in order to exclude nonspecific fluorescence due to contaminating red blood cells, a CD45 antibody labeled with PC5 (phycoerythrin-Cy5) or the like may be added. Thereafter, they were mixed gently and allowed to stand at 2-8 ° C. for 60 minutes or at room temperature for 30 minutes. After adding and stirring 1.5 mL PBS, it centrifuged for 5 minutes at 3,000 rpm. After the supernatant was aspirated and discarded, the cells were resuspended in 400 μL PBS. At this time, in order to exclude nonspecific fluorescence due to dead cells, 7-AAD viability Dye (dead cell detection reagent, MBL) may be added. Analysis was made with a flow cytometer within 24 hours.

  Confirmation of CKAP4 specific CTL was performed in two steps. First, in the first step, a portion of cells in each of the 8-wells in a 96-well U-bottom cell culture microtest plate are collected and pooled as one sample (lane pool), and the presence or absence of CKAP4 specific CTL induction It confirmed by the MHC- tetramer method. In the second step, in the lane pool in which the induction of CKAP4 specific CTL was confirmed in the first step, cells in each well were individually recovered and the presence or absence of the induction of CKAP4 specific CTL was confirmed by the MHC-tetramer method. Using such a method, it was confirmed in which well of the 96-well U-bottom cell culture microtest plate, CKAP4-specific CTL was induced.

  FIGS. 23 and 25 show representative results of the first step of confirmation after induction of CKAP4 specific CTL by the MLPC method. The PBMCs of specimen numbers A24-37 (FIG. 23) and A24-39 (FIG. 25) were cultured for 14 days with SEQ ID NO: 43 of CKAP4 specific CTL epitope candidate peptide. The numbers in the dot plot development figure are (UR + LR) when the area divided from the development figure is described as UL (upper left), UR (upper right), LL (lower left), and LR (lower right) The percentage of UR is shown. The dot plot developed view shows the fluorescence intensity for the MHC-tetramer reagent on the X-axis and the MHC-tetramer reagent on the Y-axis. The induction of a specific CTL of SEQ ID NO: 43 was confirmed by IYT-Tet, and the CD8-positive IYT-Tet-positive cells were evident in the lane 7 of lane 7 for specimen No. A24-37, and the lane 11 of A24-39. 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 specimen numbers A24-37 and A24-39. FIG. 24 and FIG. 26 show the results of the second step in which SEQ ID NO: 43-specific CTL was induced by MLPC method and detected by MHC-tetramer method. In each lane in which induction of CKAP4 specific CTL was confirmed in FIG. 23 and FIG. 25, the result of confirming presence or absence of induction of CKAP4 specific CTL in each well was shown by the MHC-tetramer method. As a result, in the G well (7-G) of sample No. A24-37 lane 7 shown in FIG. 24 and the B well (11-B) of sample No. A24-39 lane 11 shown in FIG. CTL was detected. These facts prove that SEQ ID NO: 43 is a CKAP4-specific CTL epitope peptide that exhibits HLA-A * 24: 02 restriction. Since SEQ ID NO: 43-specific CTL was detected in 1 well of 96 wells in each of specimen numbers A24-37 and A24-39, the abundance ratio of SEQ ID NO: 43-specific CTL in peripheral blood PBMC is It is calculated by the following equation.

Sample No. A24-37 SEQ ID NO: Frequency of 43-specific CTL
= (MHC-tetramer reagent positive number of wells) / (number of PBMCs used for experiment x CD8 positive rate before induction)
= 1 / (3 × 10 ⁷ × 0.311)
= 1.07 x 10 ^-7

Sample No. A24-39 SEQ ID NO: Frequency of 43-specific CTL
= 1 / (3 × 10 ⁷ × 0.155)
= 2.15 x 10 ^-7

The limit at which specific CTL can be detected by the 96-well MLPC method is usually 1.6 to 3.3 × 10 ^-7 , assuming that the percentage of CD8 positive cells before induction is 10 to 20%. In other words, in the 96-well MLPC method in this case, the standard ratio of specific CTL to be detected is 1 per 3 to 6 × 10 ⁶ CD8 positive cells.

  Furthermore, the present invention carried out the same study on seven blood donors using SEQ ID NOs: 36, 40, 42, 43 as shown in Table 8. However, detection of specific CTLs other than SEQ ID NO: 43 It was not possible. Even in SEQ ID NO: 43, only 2 out of 7 could be detected. This indicates that it is difficult to predict CKAP4-specific HLA-A * 24: 02 restricted epitope peptide, and it is more difficult to predict whether that epitope peptide has the ability to induce specific CTL. ing.

[Preparation of Antigen Presenting Cells]
Preparation of EBV-infected B cell line (Kuzushima K, Yamamoto M, Kimura H, Ando Y, Kudo T, Tsuge I, Morishima T. Establishment of anti-Epstein-Barr virus (EBV) cellular immunity by adoptive transfer of virus-specific EB95-producing cell line B95-8 cells (obtained from JCRB Cell Bank) according to cytotoxic T-lymphocytes from an HLA-matched sibling to a patient with severe chronic active EBV infection. Clin Exp Immunol. 1996; 103: 192-198) The culture supernatant (containing live EBV virus) and PBMC were mixed and cultured to establish an EBV-infected B cell line (Lymphoblastoid cell line, hereinafter referred to as EBV-infected LCL).

[Amplification of specific CTL using antigen-presenting cells]
The aforementioned antigen-presenting cells (EBV-infected LCL) are suspended in pulse medium (0.5% human serum albumin / RPMI 1640) or AIM-V medium (Invitrogen), and a CTL epitope candidate peptide is added at a concentration of 10 μg / mL. After leaving at room temperature for 30 to 60 minutes while mixing gently at intervals of approximately 15 minutes, the cells were washed three times with an excess amount of pulse medium to wash away peptides not bound 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 cells subjected to this manipulation are called peptide-pulsed antigen-presenting cells. Peptide-pulsed antigen-presenting cells were lethally treated with X-rays or mitomycin to lose their ability to proliferate. This was mixed with a cell population containing CKAP4 specific CTL derived from the same person, and cultured in a 5% CO ₂ thermostatic bath at 37 ° C. The medium used is, for example, RPMI 1640 medium containing 10% fetal calf serum (FCS), RPMI 1640 medium containing 10% human serum, or RPMI 1640 medium containing 1 to 10% human plasma, etc. In this method, Good results were obtained with RPMI 1640 medium containing 5% human plasma. IL-2 (Shionogi Pharmaceutical Co., Ltd.) was added at 50 U / mL in order to support T cell survival and support proliferation. Evaluation of CKAP4 specific CTL induction was performed 10 to 16 days after the start of culture. When induction of CKAP4 specific CTL was confirmed, peptide pulse antigen-presenting cells were further used for stimulation to establish a CTL line.

[Functional analysis of specific CTL by intracellular IFNγ-producing cell assay]
Transfer approximately 1 / 10th of the cell population containing CKAP4 specific CTL induced by the above-mentioned method to a 96-well U-bottom cell culture microtest plate, and use a final concentration of 0.1-10 μg / mL of peptide used for induction Added. Furthermore, an intracellular protein transport inhibitor (for example, Brefeldin A, Monensin etc.) was added, and the cells were cultured in a 5% CO ₂ thermostat at 37 ° C. for 5 to 16 hours. After culture, the cells were washed, PE-labeled MHC-tetramer reagent and PC5-labeled CD8 antibody (Beckman Coulter) were added, and left at room temperature for 15 to 30 minutes. After washing, the cells were fixed with 4% formaldehyde for 15 minutes at 4 ° C., and then washed with an excessive amount of washing solution. After permeabilizing with 0.1% saponin, FITC-labeled anti-IFNγ antibody (manufactured by MBL) was added, and reacted at room temperature for 15 to 30 minutes. After washing, the percentage of IFNγ positive cells in T cells or the percentage of IFNγ positive cells in MHC-tetramer reagent positive cells was quantified using a flow cytometer.

  FIG. 27 shows the results of the intracellular IFNγ-producing cell assay for CKAP4 specific CTL induced. The PBMCs of sample number A24-39 were stimulated with SEQ ID NO: 43, which is an HLA-A * 24: 02 restricted CKAP4 specific CTL epitope peptide. The results of triple staining with a PE labeled MHC-tetramer reagent (MBL), a PC5 labeled CD8 antibody, and a FITC labeled IFNγ antibody and analysis using a flow cytometer are shown. The numbers in the dot plot development figure indicate the percentage of cells present in the quadrant divided by total viable cells. FIG. 27 shows that IFNγ positive MHC-tetramer reagent positive cells appear in UR only when stimulated with a specific peptide, and hardly appear when no peptide is added. However, the presence of IYT-Tet-specific CTL in both cell populations with and without the peptide means that the X axis is CD8 and the Y axis is a log of the fluorescence intensity for the MHC-tetramer reagent. It is clear when looking at the CD8 positive MHC-tetramer reagent positive cell population in the UR in the dot plot developed view shown on the scale.

  From this result, in PBMC cultured with the CKAP4 specific CTL epitope peptide added, CKAP4 specific CTL having a cytotoxic activity to produce IFNγ is induced by restimulation, and the cells are stained with the MHC-tetramer reagent. These results revealed that the CTL was specific for the HLA-A * 24: 02-restricted CKAP4-derived peptide IYTEVRELV (SEQ ID NO: 43).

  According to the present invention, EBV infection and a virus positive cancer using a cytotoxic T cell epitope peptide specific to Epstein-Barr virus can be treated or prevented. In addition, it becomes possible to quantify CTL specific for EBV. In addition, by using an HLA-A * 24: 02 restricted epitope peptide consisting of the sequence of IYTEVRELV (SEQ ID NO: 43) of the present invention, it becomes possible to treat a disease dependent on a malignant tumor cell highly expressing CKAP4. .

Claims (16)

  A CTL epitope peptide specific for CKAP4, consisting of the amino acid sequence set forth in SEQ ID NO: 43.   A CTL epitope peptide specific for CKAP4, wherein one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 43.   HLA-A * 24: 02 molecule-restricted antigenic peptide which has a T cell receptor that specifically recognizes cells presenting a complex with the HLA-A * 24: 02 molecule on the cell surface The epitope peptide according to any one of claims 1 or 2, characterized in that it is induced.   A nucleic acid encoding the epitope peptide according to any one of claims 1-2.   An expression vector comprising a nucleic acid encoding the epitope peptide according to any of claims 1-2.   The vaccine for cancer treatment or prevention which contains the epitope peptide in any one of Claims 1-2 as an active ingredient.   A vaccine for treating or preventing cancer, comprising the nucleic acid or expression vector according to any one of claims 4 to 5 as an active ingredient.   A vaccine for treating or preventing cancer, which comprises, as an active ingredient, an antigen-presenting cell in which the epitope peptide according to any one of claims 1 to 2 is presented to HLA.   A cancer treatment comprising, as an active ingredient, the epitope peptide according to any one of claims 1 or 2 or a CKAP4 specific CTL obtained by stimulating peripheral blood lymphocytes with antigen-presenting cells presenting the epitope peptide to HLA. Immunotherapeutic agent for   A major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the epitope peptide according to any one of claims 1 to 2 and a peripheral blood lymphocyte in contact with said major histocompatibility Passive immunity for cancer treatment comprising, as an active ingredient, CTL complex obtained by forming an antigen complex and / or major histocompatibility complex-tetramer in which a CTL is bound to form a conjugate. Therapeutic agent. Stimulating peripheral blood from a subject with the epitope peptide according to any one of claims 1 to 2;
Obtaining a CTL specific for CKAP4 generated by the above steps, and measuring a cytokine and / or a chemokine and / or a cell surface molecule produced by the obtained CTL,
A method of quantifying CTL specific for CKAP4, comprising Preparing a major histocompatibility complex-tetramer from the epitope peptide according to any one of claims 1-2, and contacting the major histocompatibility complex-tetramer with peripheral blood from a subject, A method of quantifying CTL specific for CKAP4 in said peripheral blood, comprising   A method for inducing CKAP4 specific CTL, comprising the step of contacting the epitope peptide according to any one of claims 1 and 2 with peripheral blood mononuclear cells from a subject.   An epitope peptide according to any one of claims 1 to 2 or a step of stimulating peripheral blood lymphocytes with antigen-presenting cells presenting the epitope peptide to HLA to obtain CKAP4-specific CTL Method for the production of passive immunotherapeutic agents. Contacting a major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the epitope peptide according to any one of claims 1 to 2 with a peripheral blood lymphocyte,
Forming a conjugate in which CTL binds to the major histocompatibility complex and / or major histocompatibility complex-tetramer, and obtaining a CTL obtained by isolation from the conjugate. Method of producing passive immunotherapeutic agent for cancer treatment.   An antibody specific to a major histocompatibility complex prepared from the epitope peptide according to any one of claims 1-2.

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