JP5155368B2 - Cytotoxic T lymphocytes - Google Patents

Description

  The present invention relates to a method for inducing antigen-specific T lymphocytes using RNA-introduced T lymphocytes as antigen-presenting cells, and the use of the induced T lymphocytes as a therapeutic agent for cancer or infectious diseases. Further, as HLA-A2402-restricted cytotoxic T lymphocytes (CTL) specific to tumor-associated antigens MAGE-A4 or SAGE, and antigen peptides that recognize them, as well as CTL inducers and anticancer agents thereof About the use of. Furthermore, the present invention relates to a tetramer obtained by tetramerizing an MHC-antigen peptide complex useful for detecting the CTL.

  Cytotoxic T lymphocytes (CTL) include a major histocompatibility antigen MHC (human human) encoded by an antigen peptide and a major histo-compatibility gene complex (hereinafter abbreviated as MHC). A human leukocyte antigen (hereinafter abbreviated as HLA) is a complex that binds to a molecule and is recognized by a specific T cell receptor (hereinafter abbreviated as TCR). Some can damage cells presenting on the surface. Such a CTL is called HLA-restricted CTL because it recognizes and damages only target cells having the same HLA molecule as itself. Therefore, in order for the cytotoxic reaction to be established, 1) the presence of a CTL having a specific TCR, and 2) the formation of an antigen peptide that is presented to the HLA and recognized by the CTL, so that it only binds to the HLA molecule. Rather, there must be an antigenic peptide that can form a complex that is recognized by the TCR.

Such an antigen peptide is produced, for example, by processing an antigen synthesized in a mammalian cell in the endoplasmic reticulum and decomposing it into small epitope peptides, which are further associated with HLA molecules and presented on the cell surface. . That is, in a proteosome complex composed of many subunits, the protein is broken down into peptides consisting of 8 to 15 amino acids, and some of them are transported from the cytoplasm to the endoplasmic reticulum by the TAP transporter. If these peptides can bind to class I / β2 microglobulin heterodimers in the endoplasmic reticulum, they are stabilized as a trimolecular complex and transported through the Golgi apparatus to the cell surface. Tumor cells expressing tumor-associated antigens or tumor-specific antigen proteins should be able to present HLA-restricted antigen peptides that are recognized by T lymphocytes on the tumor cell surface.
Furthermore, it has been clarified that CTLs against antigens possessed by these microorganisms play an important role in infection protection even in the case of infection with viruses, bacteria, protozoa and fungi.

  HLA class I molecules mainly include HLA-A, -B, and -C, and antigen peptides that are presented by binding to these consist of 8 to 10 amino acids, and have a specific structure that differs depending on each HLA molecule. It is known that For example, the most common peptide that binds to the HLA-A2.1 molecule in the world is the peptide consisting of 9 to 10 amino acids having Leu second from the N-terminus and Leu or Val at the C-terminus. It is a known one. In addition, peptides that bind to HLA-A24 molecules, which are common in Asian races including Japanese, are Tyr, Phe, Met, Trp second from the N terminus, and Leu, Ile, Trp, Peptides consisting of 9 to 10 amino acids having any of Phe are best known [Non-patent Document 1].

  Tumor antigens for which antigenic peptides have been identified to date include MAGE-1, MAGE-3 for HLA-A1, MAGE-3 for HLA-A2.1, MART1, tyrosinase, gp100, HER2 / neu, CEA, etc. MAGE-3 for HLA-Cw1, MAGE-3 for HLA-B44, MAGE-A4 for HLA-B37, MLA-1, MAGE-2, MAGE-3, CEA, HER2 / neu, tyrosinase for HLA-A24 , Β-catenin and the like. Many of them first established a class I-restricted CTL that recognizes tumor cells, identified the tumor antigen recognized by this CTL, and subsequently found the smallest unit in the tumor antigen protein by genetic engineering methods. Furthermore, peptides in the smallest unit have been found based on information on binding motifs to HLA class I molecules [Non-patent Document 2]. In addition, based on the motif structure common to the above-mentioned HLA class I molecule-binding peptides, HLA class I molecule-binding peptides in tumor antigen proteins are found, and then those that can induce CTLs using antigen-presenting cells After selection, the antigenic peptide is determined based on whether or not CTLs that are ultimately toxic to tumor cells can be induced [Non-patent Documents 3 and 4].

On the other hand, HLA class I molecules are classified into several subtypes, but the types of possessed subtypes vary greatly among races, and HLA-A2 is the most common in the world, accounting for 45% of white races. is occupying. And identification of this HLA-A2-restricted antigen peptide is most advanced. In Japanese, HLA-A2 accounts for 40%, but looking at its subtype, HLA-A ^* 0201 is 20%, and the rest is A ^* 0206. The binding peptides to these subtypes are different, and the mainly studied HLA-A2 is HLA-A ^* 0201. On the other hand, HLA-A24 accounts for 60% or more in Japanese, and the ratio of HLA-A24 is higher in Asian races than in other races. Therefore, the discovery of HLA-A24-restricted antigenic peptides has an important role in providing CTLs useful for tumor therapy by inducing CTLs that act specifically on tumor cells in Asian races, particularly Japanese.

  Even with the same antigen, the antigen peptide differs based on the difference in HLA, and therefore induction of CTL using the antigen peptide is complicated. Various attempts have been made to solve this problem, but satisfactory results have not yet been obtained. One of the devised methods is a method for inducing T lymphocytes by transducing an antigen gene into antigen-presenting cells derived from the patient himself (self). As antigen-presenting cells, B cells, macrophages, and dendritic cells, which are known as professional antigen-presenting cells, have been studied, and clinical trials using dendritic cells as a vaccine adjuvant and the like have been conducted [non- Patent Document 5]. However, it is a problem that these antigen-presenting cells require labor to prepare an amount necessary for immunity induction. B cells can be prepared in large quantities by immortalization with EB virus, but there is a problem in safety from the point of using virus. In addition, regarding gene transfer methods, gene transfer using a viral vector or plasmid DNA cannot be denied that the gene is inserted on the chromosome and a new mutant is generated.

Journal of Immunol., 155, 4307-4312 (1995) Proceedings of the National Academy of the Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 91, pp. 3515-3519 (1994) Proceedings of the National Academy of Sciences of the USA, 91, 2105-2109 (1994) Journal of Experimental of Medicine (J. Exp. Med.), 179, 921-930 (1994) Journal of Immunotherapy, Vol. 21, pp. 41-47 (1998)

  HLA-A24-restricted antigen peptides include MAGE-1, MAGE-2, MAGE-3, CEA, HER2 / neu, tyrosinase, β-catenin, etc. in the case of tumor-associated antigens, but still not as compared with HLA-A2.1. The analysis has been delayed, and the discovery of new antigenic peptides against various tumor antigens is desired. Therefore, analysis of CTLs that act specifically on tumor cells in Asian races, particularly Japanese, has also been delayed, and it has been impossible to provide CTLs useful for tumor treatment.

  Attempts to induce CTLs using B cells, macrophages, and dendritic cells, known as professional antigen-presenting cells, have problems in handling these cells, for example, collection and expansion. Conventionally, the antigen presenting ability of T lymphocytes is not known.

  The CTL is activated by recognizing together with the MHC molecule the antigen peptide bound to the MHC molecule on the surface of the antigen-presenting cell or target cell by a complex of its cell surface T cell receptor (TCR) and the CD3 molecule, In order to cause various immune responses, analysis of CTL dynamics and functions requires the use of the cells themselves, and has been carried out using various cell lines, antigen gene transformants, etc. . However, the recently used MHC tetramer is a tetramer of the MHC-antigen peptide complex by the biotin-streptavidin method. Instead of antigen-presenting cells and target cells, CTLs having the desired TCR are also available. It has become clear that it is highly useful as a specific measurement method. However, depending on the TCR, that is, depending on the difference between the HLA and the antigenic peptide, different tetramers are required for each, and therefore, a large variety of telomers are required.

  As a result of studying a method for inducing a useful CTL against a tumor antigen, the present inventors have stimulated autologous lymphocytes with a T cell activator such as phytohemagglutinin, and electroporated RNA encoding the antigen thereto. It was clarified that the antigen-specific CTL can be induced by using the cells introduced in step 1 as antigen-presenting cells. Furthermore, HLA-A24-restricted CTL against MAGE-A4 and SAGE, which are tumor antigens, is induced, and the HLA-A24-restricted antigen peptide recognized by this CTL consists of the amino acid sequence represented by SEQ ID NO: 1 or 2 Identified as a peptide. It was clarified that this antigen peptide is useful for inducing CTL from human peripheral blood lymphocytes. At the same time, a tetramer was prepared by tetramerizing MHC-antigen peptide complex prepared using the antigen peptide of SEQ ID NO: 1 or 2 with biotin-streptavidin, and this proved useful for the detection of CTL The present invention was completed.

  A first aspect of the present invention relates to a T lymphocyte having an activity of inducing a T lymphocyte recognizing the antigen, into which RNA encoding the target antigen is introduced.

  The second aspect of the present invention relates to an immunity-inducing agent containing the T lymphocyte of the first aspect.

  A third aspect of the present invention relates to a method for inducing a T lymphocyte that recognizes the antigen, wherein the T lymphocyte of the first aspect is used as an antigen-presenting cell.

  A fourth aspect of the present invention relates to a therapeutic agent for cancer or infectious disease comprising T lymphocytes induced by the T lymphocyte induction method of the second aspect as an active ingredient.

  According to a fifth aspect of the present invention, there is provided at least one antigen peptide selected from human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof, and HLA- The present invention relates to a CD8-positive cytotoxic T lymphocyte that recognizes a cell that presents a complex with an A24 molecule on the cell surface.

  The sixth aspect of the present invention relates to an anticancer drug comprising the cytotoxic T lymphocyte of the fifth aspect as an active ingredient.

  According to a seventh aspect of the present invention, at least one antigen peptide selected from human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof is used as an active ingredient. It is related with the inducer of the cytotoxic T lymphocyte of the 5th aspect contained as.

  In an eighth aspect of the present invention, at least one antigen peptide selected from human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof is an active ingredient. It relates to an anticancer drug comprising as

  The ninth aspect of the present invention is the cytotoxicity according to the fifth aspect, comprising the human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2, and a functional derivative thereof. The present invention relates to a tetramer for detecting a T cell receptor possessed by T lymphocytes.

  According to the present invention, a method for inducing CTL using T lymphocytes that can be easily obtained as antigen-presenting cells is provided. Moreover, a novel antigen peptide restricted by (HLA) -A24 is provided. CTLs induced using the antigen-presenting cells and antigen peptides are useful, for example, in the treatment of cancer.

FIG. 2 shows the immunogenicity of peptides in HHDA2402 +/− β2m − / − mice. It is a figure which shows the result of the IFN-gamma ELISPOT assay of the CD8 + T cell derived from the spleen prepared from the mouse | mouth immunized with the gene gun using the plasmid of (a) EBNA3A cDNA, (b) MAGE-A4 cDNA, and (c) SAGE cDNA. It is a figure which shows the immunogenicity in a human of a MAGE-A4 origin peptide. (A) It is a figure which shows the result of the IFN-gamma ELISPOT assay of the human bulk CTL sensitized twice with the autologous CD4 + PHA blast cell which introduce | transduced MAGE-A4 or SAGE mRNA. Autologous LCL introduced with MAGE-A4 or SAGE mRNA was used as APC. (B) It is a figure which shows the result of having amplified well # 2 by the autologous LCL which introduce | transduced MAGE-A4 or SAGE mRNA, autologous PBMC, and IL-2 (20 IU / ml). The obtained cells were subjected to ELISPOT assay using T2A24 pulsed with a candidate peptide as APC. It is a figure which shows the result of having shown to the cell surface with HLA-A2402 after the process in the cell of the MAGE-A4 _143-151 peptide. (Left of a) MAGE-A4 _143-151 specific CTL # 2-28 cells were positive with MAGE-A4 _143-151 A24 tetramer staining (right of a) negative with control tetramer. (Left to b) This # 2-28 cell shows MAGE-A4 _143-151- specific cytotoxicity depending on HLA-A2402, and (right to b) expresses HLA-A2402 and MAGE-A4 The tumor cell line recognized MAGE-A4 _143-151 peptide processed intracellularly. It is a figure which shows the immunogenicity in human of SAGE _715-723 peptide. (A) It is a figure which shows the result of the IFN-gamma ELISPOT assay using the human bulk CTL cell obtained by sensitizing twice with SAGE mRNA introduction | transduction autologous CD4 + PHA blast cells. As target cells, T2A24 cells pulsed with SAGE _715-723 peptide or pulsed with control peptide were used. (B) It is a figure which shows the result of having expanded and cultured bulk CTL of well # 2. The obtained CTL was positive by SAGE _715-723 A24 tetramer staining. (C) IFN-γ was released only when T2A24 pulsed with SAGE _715-723 peptide was used as a target cell. It is a figure which shows the result of having shown to the cell surface with HLA-A2402 after the intracellular process of a SAGE _715-723 peptide. (A) SAGE _715-723 SAGE _715-723 specific CTL # 22 cells obtained from specific bulk CTL was positive in SAGE _715-723 A24 tetramer staining. (B) When # 22 cells (1 × 10 ⁴ cells) and SAGE cDNA-transformed 293A24 cells (1 × 10 ⁴ cells) were cultured in a 96-well round bottom plate for 18 hours, IFN-γ was released. (C) The # 22 cells were cytotoxic to K562A24 and R27A24 expressing HLA-A2402 and SAGE (left and middle in c). It also showed specific cytotoxicity against A2402-positive LCL introduced with SAGE mRNA (right of c). It is a figure which shows the result of having detected the SAGE _715-723 specific precursor in healthy person PBMC. 5 × 10 ⁵ CD8 + T cells were placed in each well and sensitized with CD8-PBMC pulsed with SAGE _715-723 peptide. A tetramer assay was performed on the 10th day, and SAGE _715-723 A24 tetramer positive CD8 + T cells were detected in 3 out of 6 people tested. FIG. 6 shows an example of the analysis result.

  A first aspect of the present invention is to induce an antigen-recognizing T lymphocyte, wherein an autologous T lymphocyte into which an RNA encoding a target antigen is introduced is used as an antigen-presenting cell (APC). On how to do.

  In the present invention, RNA encoding an antigen (target antigen) for which induction of cytotoxic T lymphocytes that specifically recognize the antigen is desired is used. The antigen is not particularly limited, and examples thereof include tumor-associated antigens and infectious microorganism antigens.

  Examples of tumor-associated antigens include SAGE, LAGE, NY-ESO-1, WT-1, and hTERT in addition to the MAGE family. Infectious microorganism antigens include EB virus antigens such as EBNA-3A, cytomegalovirus antigens such as CMVpp65, herpes virus antigens, influenza virus antigens, HIV virus antigens, Salmonella antigens, Shigella antigens, Enterobacter antigens, protozoa and fungi Examples include antigens.

  The RNA encoding the antigen can be prepared by ordinary genetic engineering techniques. Moreover, RNA prepared from cells can also be used. For example, if RNA encoding an antigen is extracted from a cell and reverse transcribed into cDNA, and this cDNA is amplified by PCR, RNA transcribed using this amplified DNA as a template can be used in the present invention. When a plasmid in which cDNA is cloned is obtained, it can be used as a template for RNA synthesis. By such a technique, it is possible to prepare a sufficient amount of RNA that can be used in the present invention even from a small amount of cells.

  There is no particular limitation on the method for artificially introducing RNA encoding the antigen into T lymphocytes used as antigen-presenting cells. Electroporation (electroporation), particle gun method, calcium phosphate method, lipofection method, liposome It can be introduced by physical methods such as law. Alternatively, RNA can be expressed in cells by introducing a gene using a viral vector such as a retroviral vector, a lentiviral vector, or an adenoviral vector.

  The T lymphocyte used as the antigen-presenting cell may be either CD4 or CD8 positive as long as it is a patient's own CD3 positive cell (autologous T lymphocyte) or a CD3 positive cell derived from a donor whose HLA type matches that of the patient. Are preferably CD4-positive T lymphocytes. In patients who have undergone transplantation such as allogeneic hematopoietic stem cell transplantation, it is also possible to use T lymphocytes of the donor of the transplant. T lymphocytes need to be activated in a medium supplemented with IL-2, IL-7, etc., using a lectin such as phytohemaglutinin (PHA), concanavalin A (ConA), or an anti-CD3 antibody. The conditions used for activation are not particularly specified as long as they are normally used. In order to use it as an antigen-presenting cell, it is treated with gamma irradiation or mitomycin.

  The T lymphocyte according to the first aspect described above can be used as an antigen-presenting cell to induce antigen-recognized T lymphocytes (third aspect of the present invention). The antigen-recognizing T lymphocytes that are induced are either CD8 positive cytotoxic T lymphocytes (CTL) or CD4 positive helper T lymphocytes, but depend on the starting material used, for example from blood Collected peripheral blood mononuclear cells (PBMC) and CD8 positive lymphocytes separated from PBMC by using anti-CD8 antibody and magnetic beads can be used, and if PBMC is used, antigen-recognizing CTL and helper T Lymphocytes usually containing both lymphocytes can be obtained. If CD8 positive cells are used, lymphocytes containing CTL can be obtained.

  When the CTL is induced in vitro, it can be induced using, for example, an in vitro extracted sample from a living body having the same type of T lymphocyte and HLA. The extracorporeal sample in this specification includes blood, lymph nodes, spleen, and other various organs removed by surgery, etc., and lymphocytes and infiltrating lymphocyte cells present in these samples are preferably used. .

  For example, when blood is used, T lymphocytes obtained by introducing the above antigen RNA into lymphocytes obtained from peripheral blood mononuclear cells (hereinafter referred to as PBMC) prepared from human blood of the same HLA type. CTL can be induced by repeatedly applying antigen stimulation with spheres. The induced CTL can be maintained as lymphocytes having stable cytotoxicity by cloning. For example, the induced CTL can be proliferated by giving a feeder cell, antigen, various cytokines, and anti-CD3 antibody stimulation.

  The activity of the induced antigen-specific CTL can be measured by damaging a target cell labeled with a radioactive substance or the like (release of the radioactive substance) after pulsing a peptide derived from the antigen that induced the CTL. It is also possible to measure the proliferation response of CTL to antigen-presenting cells pulsed with an antigen peptide by radioactivity uptake. It can be used as a target cell or antigen-presenting cell into which antigen DNA is transduced or antigen RNA is introduced. Alternatively, it can be detected by measuring the amount of cytokines such as antigen-specific GM-CSF and IFN-γ that are specifically released from CTLs and target cells. In addition, it can be directly confirmed by using an antigen peptide-HLA complex labeled with a fluorescent dye or the like. In this case, for example, CTL is contacted with a first fluorescent marker coupled with a CTL-specific antibody, and then an antigen peptide-MHC complex coupled with a second fluorescent marker is contacted, and the presence of a double-labeled cell Can be performed by FACS (fluorescence-activated cell sorting) analysis.

  Thus, the CTL induced using the T lymphocytes of the first aspect can be used as a therapeutic agent for cancer or infectious diseases (the fourth aspect of the present invention). The therapeutic agent can be formulated according to the anticancer agent of the sixth aspect described later and used for treatment.

The second aspect of the present invention relates to an immunity-inducing agent comprising the T lymphocyte of the first aspect as an active ingredient. The immunity-inducing agent is provided in the form of the T lymphocytes suspended in a pharmaceutically acceptable diluent. The diluent referred to here is, for example, a medium, physiological saline, or phosphate buffered saline suitable for storage of the T lymphocytes. Although it does not specifically limit as a culture medium, Media, such as RPMI, AIM-V, and X-VIVO10, are generally mentioned. In addition, a pharmaceutically acceptable carrier may be added to the immunity-inducing agent for the purpose of stabilization. The carrier referred to here is human serum albumin or the like. The immunity-inducing agent contains the T lymphocytes of the first aspect at 10 ⁴ to 10 ⁸ cells / ml, preferably 5 × 10 ^{5 to} 5 × 10 ⁷ cells / ml per T lymphocyte type.

The immunity-inducing agent can be administered to the T lymphocyte donor (autologous) or can be administered to another individual with a matching HLA type (allogenic). When the immunity-inducing agent is administered to humans, it can be administered, for example, subcutaneously, intradermally or intravenously with a syringe. The dose per adult is usually T lymphocytes per T lymphocyte. 10 ⁶ to 10 ¹⁰ . In addition, the said range is a standard and is not limited to this. Furthermore, the administration of the formulation can be repeated until the desired effect is obtained. The number of T lymphocytes as active ingredients is 10 ⁶ to 10 ¹⁰ . In addition, the said range is a standard and is not limited to this. In addition, since the contained cells are derived from the human being administered or have the same HLA type, toxicity of the immunity-inducing agent is not particularly observed.

  In an individual administered with the immunity-inducing agent, CTL that recognizes an antigen encoded by RNA introduced into T lymphocytes contained in the immunity-inducing agent is induced.

  In a fifth aspect of the present invention, CD8 recognizes a cell presenting a complex of the HLA-A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 or a functional derivative thereof and an HLA-A24 molecule on the cell surface. Positive cytotoxic T lymphocytes (CTL). The peptides having the amino acid sequences shown in SEQ ID NOs: 1 and 2 are MAGE-A4 and SAGE-derived HLA-A24-restricted antigen peptides, respectively. Therefore, the CTL specifically causes cell lysis or cytokine release reaction with respect to target cells or antigen-presenting cells.

  In the present specification, a functional derivative of an HLA-A24-restricted antigen peptide has an ability to form a complex with an HLA-A24 molecule, and the formed complex is represented by SEQ ID NO: 1 or 2. It is recognized by CTL that recognizes the complex of HLA-A24 molecule. For example, in the amino acid sequence of the peptide represented by SEQ ID NO: 1 or 2, one or several amino acids are deleted, substituted with other amino acids or amino acid analogs, and / or one or several amino acids or amino acid analogs The amino acid sequence varies depending on the addition or a combination thereof, but has the ability to form a complex with the HLA-A24 molecule, and the formed complex is a peptide recognized by the CTL of the present invention. The amino acid sequence length of the functional derivative is preferably 9 to 10 residues, but is not particularly limited thereto. The amino acid analog in the present specification means N-acylated products, O-acylated products, esterified products, acid amidated products, alkylated products and the like of various amino acids.

  In addition, if a complex with HLA-A24 molecule is recognized by CTL, the N-terminus and free amino group of the antigen peptide are bound to formyl group, acetyl group, t-butoxycarbonyl (t-Boc) group and the like. It may be. In addition, if the complex with HLA-A24 molecule is recognized by CTL, the C-terminus and free carboxyl group of the antigen peptide may be bonded to a methyl group, an ethyl group, a t-butyl group, a benzyl group, or the like. Good.

  The functional derivative can be identified by using CTL that recognizes a complex of the antigen peptide represented by SEQ ID NO: 1 or 2 and the HLA-A24 molecule, and examples thereof include the following methods.

  In the first method, a candidate substance as a functional derivative is mixed with HLA-A24-expressing cells, and a candidate substance unbound to the HLA-A24 molecule is washed and then reacted with CTL. If a candidate substance-specific cytotoxicity, cytokine release, or proliferative response is observed, it can be judged as a functional derivative.

  As a second method, a candidate substance is added to a cell having an antigen-presenting ability, and for an appropriate time, for example, the antigen is taken into the cell and processed, and the antigen peptide and the HLA molecule complex are presented on the cell surface. After reacting for the time required for the reaction, it is reacted with CTL. If a candidate substance-specific cytokine release or proliferation reaction is observed, it can be determined that the substance is a functional derivative.

  As a third method, a nucleic acid encoding the amino acid sequence of a candidate substance is bound to an expression vector capable of presenting a peptide on an HLA-A24 molecule on a cell having the antigen-presenting ability described later, and the recombination is performed. CTLs are reacted with cells having antigen-presenting ability transformed with a vector. If a candidate substance-specific cytokine release or proliferation reaction is observed, it can be determined that the substance is a functional derivative.

  As the functional derivative, for example, in the amino acid sequence of the peptide represented by SEQ ID NO: 1 or 2, the second amino acid from the N-terminal binds to the HLA-A24 molecule in order to strengthen the binding to the HLA-A24 molecule. Substitution with amino acids selected from Tyr, Phe, Met, Trp, which are characteristic for peptides to be selected, and / or selection from Leu, Ile, Trp, Phe, which is characteristic for peptides whose C-terminal amino acid binds to HLA-A24 molecule Among the peptides substituted with the amino acid to be bound, the complex with the HLA-A24 molecule is a complex of the peptide of SEQ ID NO: 1 or 2 and the HLA-A24 molecule. Examples include peptides recognized by CTL. For example, the peptide which substituted Phe which is the C terminal amino acid of the amino acid sequence of sequence number 2 by Leu is mentioned.

  In addition, in the peptide represented by the amino acid sequence in which one or several amino acids of the amino acid sequence described in SEQ ID NO: 1 or 2 were substituted, each substituted amino acid was substituted with an amino acid or amino acid analog having a similar side chain. Among the peptides, peptides having binding ability with HLA-A24 molecules and having complexes with HLA-A24 molecules recognized by the CTLs of the present invention are also included. Examples of amino acids having similar side chains include glycine (Gly) and alanine (Ala); valine (Val), isoleucine (Ile), leucine (Leu) and methionine (Met); asparagine (Asn) and glutamine (Gln). Aspartic acid (Asp) and glutamic acid (Glu); serine (Ser) and threonine (Thr); lysine (Lys) and arginine (Arg); phenylalanine (Phe) and tyrosine (Tyr).

  The sixth aspect of the present invention relates to a cancer drug containing the CTL of the fifth aspect as an active ingredient.

  The anticancer agent is particularly useful for the treatment of cancer in which expression of an antigen recognized by the CTL of the fifth aspect, that is, MAGE-4 or SAGE is observed.

The anticancer agent is provided in a form in which the CTL is suspended in a pharmaceutically acceptable diluent. The diluent referred to here is, for example, a medium, physiological saline, or phosphate buffered saline suitable for storing the CTL. Although it does not specifically limit as a culture medium, Media, such as RPMI, AIM-V, and X-VIVO10, are generally mentioned. In addition, a pharmaceutically acceptable carrier may be added to the anticancer agent for the purpose of stabilization. The carrier referred to here is human serum albumin or the like. The anticancer agent contains the CTL of the fifth aspect at 10 ⁴ to 10 ⁸ / ml, preferably 5 × 10 ^{5 to} 5 × 10 ⁷ / ml per CTL type.

When the anticancer drug is administered to humans, it can be administered with a syringe, and the dose per adult is usually 10 ⁶ to 10 ¹⁰ per CTL type. In addition, the said range is a standard and is not limited to this. Moreover, since the CTL which is an active ingredient is derived from the human to be administered or the HLA type is the same, toxicity of the immunity-inducing agent is not particularly recognized.

  The seventh aspect of the present invention relates to the CTL inducer of the fifth aspect, which contains the antigenic peptide of SEQ ID NO: 1 or 2 or a functional derivative thereof as an active ingredient. The CTL inducer is supplied in a form suspended in physiological saline or phosphate buffered saline as an antigen peptide alone or as a mixture with other molecules (helper T cell antigen peptide and / or adjuvant). The antigen peptide may be a covalent conjugate with a higher fatty acid or a helper T cell antigen peptide or a complex with an HLA-A24 molecule. The inducer contains 0.01 to 100% by weight, preferably 0.1 to 95% by weight, of an antigen peptide per type of antigen peptide.

  The CTL inducer can be used for in vitro diagnosis of an immunization state using, for example, an additive to a medium for growing the CTL of the present invention, T lymphocyte proliferation activity, and delayed skin reaction as an index. it can. For example, when used as an additive to the medium, the amount used is 1 ng / ml to 100 μg / ml, preferably 10 ng / ml to 1 μg / ml, as the peptide concentration in the medium. Examples of the medium generally include RPMI and AIM-V medium containing serum.

  The eighth aspect of the present invention relates to an anticancer agent containing at least one antigenic peptide selected from active ingredients of the antigenic peptide of SEQ ID NO: 1 or 2 or a functional derivative thereof. The anti-cancer agent is: 1) the antigen peptide alone, 2) a mixture of the antigen peptide and a pharmaceutically acceptable carrier and / or diluent, or 3) if necessary, supplementing 1) or 2) above. Provided in the form of an added agent. The carrier here is, for example, human serum albumin, and the diluent is, for example, phosphate buffer, distilled water, physiological saline, or the like. Examples of adjuvants include pharmaceutically acceptable adjuvants. Examples of adjuvants include (a) Freund's complete adjuvant, (b) Freund's incomplete adjuvant, (c) inorganic gels such as aluminum hydroxide and alum, (d) surfactants such as lysolecithin and dimethyloctadecyl ammonium bromide, (E) polyanions such as dextran sulfate and poly IC, (f) peptides such as muramyl peptide and tuftsin, (g) monophosphoryl lipid (MPL) A manufactured by Ribi, etc. It is not limited. When the anticancer agent is administered to humans, it can be administered, for example, subcutaneously, intradermally or intravenously with a syringe, or by percutaneous absorption from the mucosa by a method such as spraying. The anticancer agent contains 0.01 to 100% by weight, preferably 0.1 to 95% by weight, of an antigen peptide per one type of antigen peptide. The dose per adult is 0.1 μg / kg to 10 mg / kg, preferably 1 μg / kg to 1 mg / kg, more preferably 1 μg / kg to 100 μg / kg as a peptide concentration per antigen peptide. When administered to humans, toxicity of the peptide is not particularly observed.

  The ninth aspect of the present invention is the cytotoxicity according to the fifth aspect, comprising the human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2, and a functional derivative thereof. The present invention relates to a tetramer that binds to TCR of T lymphocytes. This tetramer is a complex of HLA-A24 molecule and antigen peptide prepared in the presence of β2 microglobulin and tetramerized by biotin-streptavidin method, and measured in a short time instead of CTL cytotoxicity measurement. This is useful as a simple method for measuring activity. In particular, it is useful for detection of CTL in a specimen containing only a trace amount of CTL such as PBMC and separation of CTL.

It can be used for ELISPOT (Enzyme-linked Immunospot) used for monitoring T lymphocytes in a patient's body and target cells for cytotoxicity tests.
Tetramers are useful for monitoring cytotoxic T lymphocytes.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Identification of HLA-A2402 Restricted CTL Epitope (Antigen Peptide) Using HLA-A2402 Transgenic Mice (1) Materials and Methods HHDA2402 +/− β2m − / − mice
A DNA construct (HHDA2402) consisting of the leader sequence of HLA-A2402, human β2 microglobulin, HLA-A2402 α1 and α2 domains, the transmembrane domain of H-2D ^b α3, and the cytoplasmic domain was constructed, and this was constructed using the expression vector pcDNA3. 1 (manufactured by Invirogen). The 4-kb SalI-NotI fragment of HHDA2402 was injected into eggs from pregnant C57BL / 6 mice. HHDA2402-expressing mice are crossed with β2m − / − mice (Jackson Laboratory, Bar Harbor, ME), and the resulting HHDA2402 +/− β2m +/− is crossed with β2m − / − mice to generate HHDA2402 +/− β2m − / − mice. (Hereinafter referred to as HHDA2402 mouse).

(2) Cell line A T2A24 strain was prepared by transfecting the TAP transporter-deficient strain T2 with HLA-A2402 cDNA. In addition, the following cell lines were used. Breast cancer cell line R27 (A2402 negative), esophageal cancer line KE-4 (A2402 positive), TE-10 (A2402 positive), chronic myeloid leukemia line K562 (A2402 negative), lung cancer line 11-18 (A2402 positive), Fetal kidney cells 293 (A2402 negative). R27A244, K562A24, and 293A24 were prepared by transfecting HLA-A2402 cDNA. Human B-lymphoblasts (LCL) were prepared from cells derived from HLA-A2402 positive or negative volunteers using EBV virus by a conventional method.

(3) Plasmid Full-length MAGE-A4, SAGE, and EBNA-3A cDNAs were cloned into pcDNA3.1.

(4) Immunization with gene gun Gold particles coated with plasmid DNA in the abdominal wall of HHDA2402 mice (6-8 weeks old, female) are administered with helium pressure 350-400 psi using Helios Gene Gun System (Bio-Rad) did. Gold particles were prepared according to the manufacturer's manual. A booster was administered 2 weeks later, and splenocytes were collected 1 week later.

(5) Preparation of mouse CD8 positive T cells One week after the final immunization, CD8 positive cells were positively selected by MACS system (Miltenyi Biotec, Germany) using CD8 alpha (Lyt2) microbeads. The obtained T cell fraction was 95% or more pure by flow cytometry analysis.

(6) ELISPOT assay (mouse)
A 96-well nitrocellulose ELISPOT plate (MAHA S4510; Millipore, Bedford, MA) was coated overnight at 4 ° C. with 2 μg / ml anti-mouse IFN-gamma mAb (clone R4-6A2, PharMingen). Each well was washed with phosphate buffered saline (PBS) and then blocked with a medium containing FCS (37 ° C., 2 hours). Separated fresh CD8-positive cells (1 × 10 ⁵ / well) derived from immunized mice and CD8-negative cells (1 × 10 ⁶ / well) pulsed with various peptides were seeded in each well so that the final volume was 200 μl. . After culturing at 37 ° C. for 22 hours, thoroughly washed with PBS containing 0.05% Tween 20 (PBS-Tween), added with 1.25 μg / ml biotinylated anti-mouse IFN-gamma mAb (PharMingen), and cultured at 4 ° C. overnight. did. After washing with PBS-Tween, 100 µl of 1 µg / ml streptavidin-alkaline phosphatase conjugate (Mabtech) was added, reacted at room temperature for 90 minutes, washed with PBS-Tween, and stained with alkaline phosphatase conjugate substrate kit (BioRad, Hercules, CA) . Then, the reaction was stopped by washing with distilled water, the plate was then dried, and spots were counted.

(7) Epitope peptide estimation A 9-residue peptide that may have HLA-A2402 binding ability is obtained from the BioInformatics & Molecular Analysis Section (BIMAS) HLA Peptide Binding Prediction website (URL; http: //bimas.dcrt.nih). .gov / molbio / hla-bind /). Five SAGE-derived peptides listed in Table 1 below and 10 peptides derived from MAGE-A4 were selected and chemically synthesized using a peptide synthesizer. In addition, EBNA3A _246-254 (RYSIFFDYM) derived from EB virus and HER2 _63-71 (TYLPTNASL) derived from HER2 were synthesized. The purity of the peptide used is 90% or more.

HHDA2402 mice were immunized with a gene gun with an expression plasmid encoding the EBNA3A gene derived from EB virus. After immunization twice, CD8 + T cells derived from spleen were prepared, and ELISPOT assay was performed using CD8 negative cells pulsed with the EBNA3A _246-254 peptide as target cells. As a result, EBNA3A _246-254 peptide-specific CTL was detected (FIG. 1a). Next, ELISPOT assay was similarly conducted on candidate peptides derived from MAGE-A4 and SAGE (Table 1 above), which are tumor / testis antigens. As a result, two peptides (MAGE-A4 _239-247 and MAGE-A4 _143-151 ) were positive in MGE-A4 (FIG. 1b). Similarly, two peptides (SAGE _776-784 and SAGE _715-723 ) were positive in SAGE (FIG. 1c). Next, when wild-type C57BL6 mice were _subjected to the same experiment as described above, only MAGE-A4 _239-247 and SAGE _776-784 were positive. Therefore, MAGE-A4 _143-151 and SAGE _715-723 were identified as antigenic peptides presented to HLA-A2402.

Example 2 Preparation of HLA-2402 Antigen Peptide Specific Human Cytotoxic T Lymphocytes (CTL) Using CD4-Positive PHA Seed Cells Introduced with mRNA (1) Preparation of mRNA MAGE-A4 and SAGE plasmid were linearized I made it. In vitro transcription was performed according to the manufacturer's manual using T7 polymerase (mMESSAGE mMACHINE T7 Kit; Ambion, Austin, TX). Subsequently, polyadenylation was performed using poly A polymerase (Poly (A) Tailing Kit; Ambion, Austin, TX) according to the manufacturer's manual. The obtained RNA was suspended in water and stored at −80 ° C. until use.

(2) Preparation of CD4 positive phytohemagglutinin (PHA) blast cells Fresh CD4 positive cells obtained by separation by positive selection using MACS CD4 microbeads (Miltenyi Biotec, Germany) Corning, NY) cells at 1-2 × 10 ⁶ cells / ml RPMI-1640 medium (25 mM Hepes, 10% inactivated human AB serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin) per well. Planted in concentration. On Day 0, 10 μg / ml PHA (HA15, Murex, UK) was added to the medium. On Day 3, half of the medium was replaced with complete medium containing IL-2 (20 U / ml) and IL-7 (40 ng / ml). The same operation was repeated every 3 days. The obtained activated CD4 positive cells were electroporated 14 to 28 days after culture and used as antigen-presenting cells.

(3) In vitro human CTL induction using mRNA-introduced CD4 positive blast cells 5 × 10 ⁵ CD8 + T cells isolated from PBMC using MACS CD8 Microbeads (Miltenyi Biotec) are 1 × 10 ⁵ radiation. (30 Gy) Irradiated mRNA-introduced CD4 + PHA blast cells were stimulated in 96-well round bottom plates (Nunc). Seven days later, CD8 + T cells were restimulated with mRNA-introduced CD4 + PHA blast cells irradiated with 1 × 10 ⁵ radiation (30 Gy), and further cultured in a medium containing IL-2 (20 IU / m) for 7 days.

(4) In vitro amplification of CTL In order to amplify sensitized CD8 + T cells containing antigen-specific CTL, self-LCL (5 × 10 ⁶ cells) into which target antigen mRNA was introduced by electroporation, self-PBMC (2. 5 × 10 ⁷ ) and IL-2 (20 IU / ml) were added and cultured in a 25 ml flask (Corning) without anti-CD3 antibody.

(5) Limiting dilution Dilute to 96 / well round bottom plate (Nunc) to 0.3 / well, autologous PBMC (5 × 10 ⁴ / well), irradiation LCL (1 × 10 ⁴ / well) ), IL-2 (20 IU / ml), and anti-CD3 mAb (30 ng / ml). CD8 + T cells that lyse target antigen-presenting cells were further amplified in the presence of irradiated PBMC, irradiated LCL and anti-CD3 mAb.

(6) ELISPOT assay (human)
A 96-well nitrocellulose ELISPOT plate (MAHA S4510; Millipore, Bedford, MA) was coated overnight at 4 ° C. with 2 μg / ml antihuman IFN-γ mAb (1-D1K). Each well was washed with PBS and then blocked with a medium containing 10% human AB serum (37 ° C., 2 hours). Effector cells (2 × 10 ⁴ / well) and peptide-pulsed T2A24 cells (5 × 10 ⁴ / well) were seeded in each well. After culturing at 37 ° C. for 18 hours, the plate was thoroughly washed with PBS-Tween, added with 1.25 μg / ml biotinylated anti-mouse IFN-gamma mAb (PharMingen), and cultured at 4 ° C. overnight. After washing with PBS-Tween, 100 μl of 1 μg / ml streptavidin-alkaline phosphatase conjugate (Mabtech) was added, reacted at room temperature for 60 minutes, washed with PBS-Tween, and then stained with alkaline phosphatase conjugate substrate kit (BioRad, Hercules, CA). . Then, the reaction was stopped by washing with distilled water, the plate was then dried, and the number of spots was counted.

(7) ⁵¹ Cr free cytotoxicity assay Cytotoxicity was performed according to a conventional method. Target cells were labeled with 100 μCi (3.7 × 10 ⁶ Bq) ⁵¹ Cr, and 1 × 10 ⁴ cells were reacted at 37 ° C. with effector cells having different cell numbers in a 96-well V-bottom plate (Nunc). After 5 hours, 100 μl of supernatant was collected and measured. The assay was performed in triplicate and the average% specific lysis for 3 wells was calculated.
Specific cytotoxic activity (%) = [(measured value of each well−minimum release value) / (maximum release value−minimum release value)] × 100
In the above formula, the minimum release value is the amount of ⁵¹ Cr in a well containing only target cells and K562 cells, and indicates the spontaneous release amount of ⁵¹ Cr from the target cells. Further, the maximum release value indicates the ⁵¹ Cr release amount when the cell is destroyed by adding the surfactant Triton X-100 to the target cells.

CD8 + cells prepared from healthy subjects were sensitized with autologous CD4 + PHA blasts into which mRNA was introduced in vitro. MAGE-A4 specific bulk CTLs were obtained after two stimulations with autologous CD4 + PHA blasts transfected with MAGE-A4 mRNA (FIG. 2a). Cells obtained by amplifying this bulk cell with mRNA-introduced autologous LCL, autologous PBMC and IL-2 showed a reaction specific to T2A24 cells pulsed with MAGE-A4 _143-151 peptide (FIG. 2b). The # 2-28 cells obtained by the limiting dilution method were positively stained with the MAGE-A4 _143-151 tetramer, but were not stained with the tetramer used as a target (FIG. 3a). The # 2-28 cells showed HLA-A2402-restricted cytotoxicity to the target cells pulsed with the MAGE-A4 _143-151 peptide (left of FIG. 3b). Furthermore, the 2-28 cells showed cytotoxicity against tumor cell lines expressing both MAGE-A4 and HLA-A2402 (right side of FIG. 3b). This indicates that the MAGE-A4 _143-151 peptide is processed in the cell and presented as an antigen not only in mRNA-introduced CD4 + PHA blast cells but also in HLA-2402-positive tumor cells.

Next, SAGE _715-723- specific bulk CTLs were induced in CD4 + blast cells transfected with truncated SAGE mRNA by stimulation twice (FIG. 4a). Flow cytometric analysis revealed that this bulk CTL _contained SAGE _715-723 HLA-A24 tetramer positive CD8 + cells (FIG. 4b). SAGE _715-723- specific HLA-A24 CTL cells # 22 were prepared by the limiting dilution method. The # 22 cells were SAGE _715-723 HLA-A24 tetramer positive (FIG. 5a). This cell secreted IFN-γ when co-cultured with 293-A2402 gene-transfected with a plasmid encoding the full-length SAGE gene (FIG. 5b). The cells were cytotoxic to both K562A24 and R27A24 (both expressing HLA-A2402 and SAGE) (FIG. 5c left and middle). Furthermore, it showed specific cytotoxicity against A2402 + LCL cells into which mRNA of the SAGE gene was introduced (right of FIG. 5c).

Example 3 SAGE _715-723- specific CD8 + T cells are induced with high probability from A2402-positive healthy individuals.
Induction of human CTL using peptide-pulsed CD8-PBMC in vitro 1 × 10 ⁷ CD8-negative PBMCs were pulsed with 10 μM peptide for 1 hour at room temperature and 37 ° C. with 5% CO ₂ for 1 hour. Later, it was used as an antigen-presenting cell. The isolated 5 × 10 ⁵ CD8 + T cells were then stimulated with 1 × 10 ⁶ CD8-PBMC pulsed with the peptide for 10-12 days. On days 1, 4, and 7, half of the medium was changed and human IL-2 (20 IU / ml) and IL-7 (50 ng / ml) were added. Induction was performed in 200 μl RPMI medium (25 mM Hepes, 10% inactivated human AB serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin) in a 24-well (Nunc) plate.

HLA-A2402-restricted SAGE _715-723- specific CTL is induced by stimulating CD8 + cells once in vitro with CD8-negative PBMC pulsed with SAGE _715-723 peptide in healthy volunteers positive for HLA-A2402 We examined what to do. SAGE _715-723 HLA-A24 tetramer positive T cells were detected 10 days after in vitro mixed lymphocyte reaction in 3 out of 6 HLA-A2402 + healthy subjects. FIG. 6 shows an example of the analysis result.

Example 4 Preparation of tetramer and flow cytometry analysis
For the preparation of MHC-peptide tetramer, HLA-A2402 heavy chain and β2-microglobulin were expressed in E. coli as insoluble polymers. A sequence serving as a substrate for the biotinylation enzyme BirA was added to the C-terminus of the heavy chain. _Monomeric HLA / β2-microglobulin / peptide complexes were folded in vitro in the presence of MAGE-A4 _143-151 peptide or SAGE _715-723 . MHC was biotinylated with BirA enzyme recombinant (Avidity, Denver, CO, USA) and tetramerized using phycoerythrin-labelled streptavidin (Molecular Probes, Eugene, OR, USA).

  For staining, sensitized CD8 + T cells were reacted with tetramer 20 μg / ml at 37 ° C. for 30 minutes, and then reacted with Tricolor anti-CD8 monoclonal antibody (Caltag, Burlingame, Calif., USA) for 15 minutes on ice. After washing, the stained cells were analyzed by flow cytometry (FACSCalibur; Becton Dickinson).

In addition, the following are mentioned as an aspect of this invention.
[1] A T lymphocyte having an activity of inducing a T lymphocyte recognizing the antigen, into which an RNA encoding the target antigen is introduced.
[2] The T lymphocyte according to [1] above, wherein the T lymphocyte is a CD4 positive cell activated with phytohemaglutinin.
[3] An immunity-inducing agent containing the T lymphocyte according to [1] or [2].
[4] A method for inducing T lymphocytes that recognize antigens, wherein T lymphocytes into which RNA encoding a target antigen is introduced are used as antigen-presenting cells.
[5] The induction method according to [4], wherein the T lymphocyte is a CD4-positive cell activated with phytohemaglutinin.
[6] The induction method according to [4], wherein the T lymphocyte recognizing an antigen is a CD8 positive cytotoxic T lymphocyte.
[7] The induction method according to any one of [4] to [6], wherein the antigen is a tumor-related antigen or an infectious microorganism antigen.
[8] The induction method according to [7] above, wherein RNA prepared from cancer tissue is used.
[9] The induction method according to [7], wherein RNA expressing at least one viral antigen of EBNA3A and CMVpp65 is used.
[10] A therapeutic agent for cancer or infectious disease comprising T lymphocytes induced by the method according to any one of [4] to [9] as an active ingredient.
[11] A complex of at least one antigen peptide selected from human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof and an HLA-A24 molecule CD8-positive cytotoxic T lymphocytes that recognize cells that present on the cell surface.
[12] An anticancer agent comprising the cytotoxic T lymphocyte according to [11] as an active ingredient.
[13] A human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and at least one antigen peptide selected from functional derivatives thereof as an active ingredient, The inducer of cytotoxic T lymphocyte according to [11] above.
[14] A system comprising at least one antigen peptide selected from the human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof as an active ingredient. Cancer drug.
[15] The cytotoxic T lymphocyte according to [11] above, which contains the human major histocompatibility antigen (HLA) -A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2 and a functional derivative thereof Tetramer for T cell receptor detection.

  Since the method for inducing antigen-recognizing T lymphocytes of the present invention can induce specific T lymphocytes against antitumor or anti-infectious microbial antigens regardless of HLA, the induced specific T lymphocytes are cancer or It is useful as a therapeutic agent in immunotherapy for infectious diseases. In addition, HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) specific to MAGE-A4 or SAGE, which are tumor-associated antigens, and antigen peptides that recognize them are useful as anticancer agents in immunotherapy. Furthermore, the MHC-antigen peptide complex tetramer using the antigen peptide is useful for measuring the function of the prepared CTL and monitoring after CTL administration.

Claims (5)

Human major histocompatibility antigens of SEQ ID NO: 2 (HLA) -A24 restricted antigen peptide and HLA-A24 complex with molecules of cells recognizing CD8 positive presented on the cell surface cytotoxic T lymphocytes ball.   An anticancer agent comprising the cytotoxic T lymphocyte according to claim 1 as an active ingredient. Comprising as an active ingredient the human major histocompatibility antigens (HLA) A24 restricted antigen peptide represented by SEQ ID NO: 2, inducer of cytotoxic T lymphocytes according to claim 1. Human major histocompatibility antigens (HLA) A24 anticancer agent comprising a restricted antigen peptide as an active component represented by SEQ ID NO: 2. Containing human major histocompatibility antigens (HLA) A24 restricted antigen peptide represented by SEQ ID NO: 2, tetramer for T cell receptor detection with the cytotoxic T lymphocyte according to claim 1, wherein.

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