TW201245224A - Cytotoxic t cell inducing composition - Google Patents

Abstract

The present invention develops a novel technology that is able to easily perform antigen-specific CTL induction for any given antigen. The present invention provides a cytotoxic T cell inducing composition containing: an anti-CD28 antibody; a solid support body at which the anti-CD28 antibody has been solid-phased; and a soluble peptide that can bond to an MHC class I molecule. In the cytotoxic T cell inducing composition, there are cases of the soluble peptide that can bond to an MHC class I molecule being presented as an antigen by means of an HLA compliant HLA complex of the patient and being recognized by the cytotoxic T cells. The present invention provides a tumor treatment drug composition. The tumor treatment drug composition contains the cytotoxic T cell inducing composition, the soluble peptide that can bond to an MHC class I molecule contains a portion of the amino acid sequence of a specific antigen protein of tumor cells, and the cytotoxic T cells recognize the tumor cells. The specific antigen protein of the tumor cells can be WT-1.

Description

201245224 VI. Description of the Invention: [Technical Field] The present invention relates to a composition for inducing cytotoxic tau cells, and a cytotoxic T cell obtained by exposing the composition for inducing cytotoxic tau cells A pharmaceutical composition, and a method of producing the pharmaceutical composition. [Prior Art] In vivo, antigen-specific cytotoxic T cells (hereinafter also referred to as "CTL") are induced by interaction between CD8-positive cells derived from hematopoietic stem cells and dendritic cells. The aforementioned interaction involves a complex between the fragment of the antigen molecule and the surface HLA class I molecule of the dendritic cell, and the aggregation of the TCR/CD3/CD8 complex on the surface of the CD8 positive cell. And 'as a result of the aforementioned interaction, the aforementioned CD8-positive cells will become a complex that specifically recognizes and attacks the HLA class I molecules consistent with the compatible Hla class I molecules and fragments of the aforementioned antigen molecules. The cells. The aforementioned interaction, in addition, is also promoted by the aggregation of the surface CD28 of the CD8-positive cells and the CD80/86 molecules on the surface of the dendritic cells. In the case of viral infection, cytotoxic T cells that specifically recognize viral antigens attack the virus-infected cells. In the case of tumor cells, cytotoxic tau cells that recognize tumor-specific antigens attack tumor cells. How to efficiently induce cytotoxic Τ cells in tumor immunotherapy becomes a problem. Due to advances in the study of cytokine (cyt〇kine), it has become easier to differentiate from hematopoietic stem cells such as gallbladder blood, terminal blood, and bone marrow, and to proliferate cytotoxicity to CD8 positive cells of cells before the 201245224 cell. As a result, it is difficult to refine the dendritic cells from a large number of living bodies. Therefore, techniques for artificially mimicking the function of dendritic cells have been developed. An example of the artificial antigen-presenting cell is a technique in which a gene such as an HLA diterpenoid molecule, an antigen molecule, or a CD8〇/86 molecule is transfected into a cultured cell such as a fibroblast, and a dendritic cell function is performed. Further, a complex of an MHC class I molecular quaternary body and an antigenic peptide (hereinafter referred to as an "antigen quaternary body") has a high dissociation constant and can be detected because it can bind to most T cell receptors on the same cell surface. In addition to T cells which recognize a specific antigen (Non-Patent Document 1), activation of antigen-specific T cells is also used (Non-Patent Document 2). Further, phycoerythrin (hereinafter referred to as "pe") ") labeled antigenic quaternary and PE-labeled anti-CD28 antibody, which can induce cytotoxic T cells via fluorescently labeled paramagnetic beads bound by biotinylated anti-pE antibody and avidin (non-patented) Document 3). Although it cannot be called a living organism, it is an artificial antigen-promoting cell. There are also techniques for binding molecules on the surface of beads, microparticles, etc. against CD8-positive molecules, instead of dendritic cells to stimulate CD8-positive cells. The technique of using a solid phase antibody is known as an anti-CD3/CD28 bead which is obtained by binding an anti-CD3 antibody having an agonist activity and an anti-CD28 antibody to the same bead (Patent Document 1) ). In the interaction of induced CTL, the complex between the fragment of the antigen molecule and the HLA class I molecule on the surface of the dendritic cell, and the aggregation of the TCR/CD3/CD8 complex on the surface of the CD8 positive cell can be used by using The agonist-active anti-CD3 antibody replaces the antigen molecule fragment with the HLA class I molecule on the surface of the dendritic cell. 201245224 - Body, stimulates CD8-positive cells. Also, on the surface of CD8-positive cells, CD28 is sub- and dendritic The aggregation of CD80/86 molecules on the cell surface can be used to replace CD80/86 on the surface of dendritic cells by using an anti-CD28 antibody with agonist activity, but it is only resistant to CD3/CD28 beads. In order to induce antigen-specific CTL, in order to induce antigen-specific CTL, it is necessary to use a living-derived antigen-presenting cell or an artificial antigen to present a cell. [Prior Art] Patent Document 1: International Publication No. WO 2004/1 041 Book No. 85 Non-Patent Document Non-Patent Document 1: Altman, jD et al. 'Science, 274: 94 (1996) Non-Patent Document 2: Daniels, Μ·Α· and James〇n, s c., j.

Exp. Med., 191: 335 (2000) Non-Patent Document 3: Oelke, M. et al., Nature Medicine, 9:6 1 9 (2003) ' [Summary of the Invention] (The problem to be solved by the invention) is to make a habit Knowing that the antigen indicates that the cell or the artificial antigen indicates that the cell needs time and labor, and it is necessary to develop a novel technique for easily inducing antigen-specific CTL for any antigen. ', (the way to solve the problem) The present invention provides For the composition for inducing cytotoxic T cells, 5 201245224 comprises an anti-CD28 antibody, a solid support having the anti-CD28 antibody immobilized, and a soluble conjugate which can bind to the MHC class I molecule. The composition of the cytotoxic T cell may be composed of an anti-CD28 antibody, a solid support having the anti-CD28 antibody immobilized, and a soluble peptide capable of binding to the MHC class I molecule. In the composition for inducing cytotoxic tau cells, the solid support may be a culture vessel or microbeads for culturing cells containing the aforementioned CD8-positive cells. The composition of the cell, the soluble peptide capable of binding to the (4) 帛! molecule, can be recognized by the HU compatible (four) 彪 complex of the patient as an antigen, which is recognized by the cytotoxic tau cell. A pharmaceutical composition for treating a tumor. The pharmaceutical composition for treating a tumor of the present invention comprises a composition for inducing cytotoxic sputum cells of the present invention, and the aforementioned soluble peptide capable of binding to a steroid class I molecule comprises The partial amino acid sequence of the specific antigenic protein of the tumor cell 'the aforementioned cytotoxic sputum cell identifies the aforementioned tumor cell. The specific antigenic protein for treating the tumor cell of the present invention may be a pharmaceutical composition, the aforementioned tumor fine WT -1. The present invention provides a pharmaceutical composition for cytotoxic butyroxic disease in which a soluble peptide comprising a pharmaceutically acceptable cytotoxic tau cell for treating a viral disease is combined with a virulence peptide. The composition 'containing the partial amine cell of the present invention for tempting' and the aforementioned specific antigenic protein capable of interacting with MHC class I molecules 201245224 In the pharmaceutical composition for treating a viral disease of the present invention, the specific antigenic protein of the virus may be a pp65 protein of cytomegalovirus. The present invention provides a pharmaceutical composition. The pharmaceutical composition of the present invention comprises the present invention. The composition for inducing cytotoxic tau cells of the invention is exposed to cytotoxic tau cells obtained from CD8 positive cells derived from hematopoietic stem cells, and the cytotoxic tau cells recognize the aforementioned soluble peptide capable of binding to MHC class I molecules. In the pharmaceutical composition of the present invention, the hematopoietic stem cell may be selected from the group consisting of embryonic stem cells, adult stem cells, and artificial buds (i PS) cells. Any of the stem cell-derived hematopoietic stem cells, cord blood-derived hematopoietic stem cells, peripheral blood-derived hematopoietic stem cells, and bone marrow blood-derived hematopoietic stem cells. The present invention provides a method of producing a pharmaceutical composition comprising cytotoxic T cells. The method for producing a cytotoxic T cell-containing pharmaceutical composition of the present invention comprises the steps of: (1) proliferating a CD8-positive cell from at least one hematopoietic stem cell selected from the group consisting of: Embryonic stem cells, adult stem cells, and artificial pluripotent stems (groups composed of phantom cells, hematopoietic stem cells derived from any stem cells, hematopoietic stem cells derived from cord blood, hematopoietic stem cells derived from peripheral blood, and bone marrow Blood-derived hematopoietic stem cells; (2) exposing the composition for inducing cytotoxic tau cells as described in (1) to the aforementioned CD8-positive cells; (3) culturing and identifying the above-mentioned soluble salt capable of binding to MHC class I molecules 7 201245224 Peptide cytotoxic tau cells. In the method for producing a pharmaceutical composition comprising cytotoxic tau cells of the present invention, the aforementioned soluble peptide capable of binding to MHC class I molecules may comprise human WT-1 protein, or giant cells. Amino acid sequence of the ρρ65 protein of the virus. The present invention provides an immunotherapy comprising transplanting a pharmaceutical composition comprising the cytotoxic sputum cell of the present invention. The immunotherapy of the present invention may comprise the steps of: CD8 positive cells obtained by taking hematopoietic stem cells of the patient's terminal blood and bone marrow, but not limited thereto, or CDs obtained by differentiating and/or proliferating the obtained hematopoietic stem cells. The step of exposing 8 positive cells 'in vitro to the composition for inducing cytotoxic sputum cells of the present invention' and the step of re-implanting the induced cytotoxic sputum cells into the patient. The immunotherapy of the present invention may include the following Step: Exposing CD8-positive cells obtained from a patient's somatic cell-derived adult stem cells or from a patient's somatic cell-derived artificial pluripotent stem cells to an in vitro test for inducing cytotoxic sputum cells The step of the composition, and the step of re-transplanting the induced cytotoxic sputum cells into the patient. The source of the CD8 positive cells of the present invention comprises terminal blood, bone marrow, lymph nodes and cord blood 'but may not be limited to the tissue described herein. The dendritic cells of the invention or their precursor cells are preferably prepared from the final blood. However, from embryonic stem cells, adult stem cells and artificial pluripotent It is also possible to prepare hematopoietic stem cells produced by stem cells (iPS). In order to induce CD8-positive cells into cytotoxic tau cells, it is necessary to use MHC class J molecules and antigenic antigens while puncturing CD28 on CD42 positive cells in 201245224. The antigen complex is expressed by the aggregation of the complex with the tau cell receptor/cd3/cd8 complex. According to the present invention, only the solid phase anti-CD28 antibody and the soluble antigen can be used, and no dendritic cells are used. CD8-positive cells are induced into cytotoxic tau cells. The mechanism of action is not limited to theory, but the invention can be reasonably explained by inferring the participation of cells expressing Hu class I molecules when culturing CD8-positive cells. The action of the composition of cytotoxic T cells is operative. The figure is used to illustrate the schematic diagram of the action of the present invention. Referring to Fig. i, CD8-positive cells were induced into CTLs by soluble peptide 2, and anti-CD28 antibody 4 immobilized on solid support 3 such as magnetic beads. Here, the anti-CD28 antibody 4' immobilized on the solid support 3 will react with the CD28 molecule 5 on the CD8-positive cell 1, and the CD8-positive cell 1 will provide the same interaction with the CD80/86 on the dendritic cells. stimulate. On the other hand, only soluble peptide 2 was not recognized by the tau cell receptor/CD3/CD8 complex 6 on CD8 positive cells 1. However, when the soluble peptide 2 is bound to the HLA class I molecule 7 on the cell 8 expressing the HLA class I molecule in the CD8 positive cell 1 culture, it will be used as an antigen to prompt the T cell on the CD8 positive cell 1. Body/CD3/CD8 complex 6. Thus, only the soluble peptide 2 and the anti-CD28 antibody immobilized on the solid support 3 can be added to the CD8 positive cells 1 to induce the recognition of the HLA class I molecule 7 from the CD8 positive cells 1 2 cytotoxic T cells 9 . According to the method of the present invention, CTL induction can be performed on different kinds of antigens by merely replacing the soluble peptide. At the same time, CTL induction can be performed on two or more antigens. 201245224 The invention can hide and conceal! The soluble peptide which is a molecule-like molecule can be composed of the amino acid sequence listed in the sequence of the sequence listing attached to the present specification. The compensable peptide of the present invention which binds to a ?-like molecule is not limited to the HU-A restricted epitope. It has been known from the past that cells which are capable of proliferating in vitro can be cytotoxic to other antigens, and that clinical effects can be obtained by transplanting cytotoxic T cells into patients. For example, Gode U. et al. obtained a tyrosinase-derived HLA_B-restricted peptide derived from melanoma-specific cancer antigen-specific cytotoxic tau cells (Cancer Im_〇1. Immun〇th^. , 58:271 -280 (2009)). Stratahof, KCM et al. HLA-B restriction epitope of 杬原LMP2 associated with other viruses, HLA-B restriction epitope of EB virus-associated antigen EBNA1, HLA-B of antigen EBNA3 associated with Εβ virus Restriction epitope, Epstein-Barr virus-associated antigen BZLF1 HLA-B-restricted epitope is a specific CTL, which is transplanted into Epstein-Barr virus-associated nasopharyngeal carcinoma patients to obtain clinical results (Blood, 1 05:1 898 -1 904 (2005 )). Walter, EA et al., after transplantation of bone marrow from CMV-positive blood donors for the treatment of leukemia, during the administration of immunosuppressive agents, cytomegalovirus (CMV)-specific CTLs were transplanted to reactivated patients. Clinical effect (N. EngL j.

Med., 333:1 038-44 (1995)). 'Bi〇iey G. et al., obtained cytotoxic tau cells specific for the antigen-derived HLA-B or HLA-C-restricted peptides from patients who have been vaccinated with the cancer antigen NY-ES0-1 vaccine ( Clin. Cancer Res. 1 5:299-306 (2009)). However, the soluble peptide which can bind to the MHC class I molecule of the present invention can be not only the HLA_A restriction epitope such as the white matter source peptide of the town-tuck egg 201245224, but also HLA-B or HLA-C. Restriction epitope. The disease-associated antigen expressed by the patient's cells may include WT-1, human granulocyte reverse transcriptase (hTERT), survi vin, tyrosinase, Νγ-ESO-i, CEA, NSE, PSA, gPi〇〇, MART-1 and MAGE-3, but not limited to such tumor-associated antigens, antigens expressed in cancer cells (over) such as Epstein-Barr virus-associated antigens such as EBER, LMP-1, may include cytomegalovirus Pp65 (CMVpp65) An cytomegalovirus-specific antigen such as a protein, an HIV-specific antigen such as HlVgpi6〇, but not limited to an antigen expressed by an infected cell such as a virus-specific antigen. Therefore, the medical group of the present invention can be used for the treatment of cancer and infectious diseases. The soluble peptides of the present invention which bind to the 〇c class I molecule may also be epitopes of such antigens. The solubility of the present invention which binds to the MHC class I molecule can be composed of the same amino acid sequence as the native protein or an amino acid sequence different from the amino acid portion of the native protein. Alternatively, it may be a peptide containing an amino acid sequence identical to the native protein or an amino acid sequence different from the amino acid portion of the native protein. For example, the aforementioned soluble peptide sometimes consists of amino acid sequences 126 to 134 of the WT-1 protein (SEQ ID NO: 1), and amino acids 235 to 243 of WT-1 protein. A mutant amino acid sequence (SEQ ID NO: 2) obtained by substituting a thiol acid residue of No. 236 in the sequence for a tyrosine residue, and an amino acid of No. 461 to No. 469 of hTERT Sequence (SEQ ID NO: 3), amino acid No. 80 to No. 88 of the segmented variant 2B of Survivin 201245224, second sequence identification number 4), and 34i to _ number of CMVpp65: amine:: The amino acid sequence selected from the group consisting of the sequence (SEQ ID NO: 5) consists of, or contains, an amino acid sequence selected from the group. The solid phase anti-CD28 fossa of the present invention may be any antibody having agonist activity for CD28. For solid phase, any solid support capable of performing ctl induction can be used which is close to the surface of the (10) positive cells. The preferred solid support of the present invention is decent, comprising latex, other commercially available microparticles and culture capacity, and is but not limited to. The culture vessel includes a culture dish, a flask, a plate, and a double liter plate, but is not limited thereto. The anti-(10)8 antibody of the present invention may be directly bound to the solid support of the present invention, or may be subjected to a specific interaction such as azine, biotin or the like. The combination of special-sexual partners. Microbeads immobilized with streptavidin and biotinylated anti-CD28 antibodies can also be used. The aforementioned microbeads may have magnetic properties. Hereinafter, an anti-CD28 antibody immobilized on a microbead, which is called an anti-CD28 immunobead, will be immobilized using a specific-sex interaction of biotin and streptavidin. The pharmaceutical composition 'of the present invention' may contain a carrier which is acceptable as a pharmaceutical. Representative examples are any solution capable of suspending living cells, such as physiological saline, phosphate buffered saline (PBS), culture medium, serum, and the like. [Embodiment] (Embodiment of the Invention) The embodiments of the present invention described below are merely illustrative and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the scope of the patent application. The present invention can be modified, for example, by adding, deleting, and replacing the constituent elements of the present invention without departing from the scope of the present invention. Example 1 Induction of WT-1 peptide specific CTL from cord blood (1) 1. Materials and methods (1) Human cord blood The human cord blood system was informed by the mother and agreed to sign the blood, frozen and stored in the Institute of Physical Chemistry. Inside the liquid nitrogen system in the Bioresorce center. The blood draw method is described in Rubinstein, P. et al., Blöd, 81,: 1 679-1 690 (1 993), cryopreserved in Rubinstein, P et al., Proc. Natl. Acad. Sci. USA, 92 :1 0 1 1 9-1 0 1 22( 1 995) Description. (2) Medium and reagents The medium was X-VIV0 (trademark) 15 (Takarabio Co., Ltd., Shiga). In some experiments, human AB-type serum (Lonza, Lonza Japan Co., Ltd.) with a final concentration of 〇~5% was added. Recombinant human IL-7 (Peprotech, Toyobo Co., Ltd.) and IL-1 5 (Peprotech, Toyobo Co., Ltd.) were each added at a final concentration of 0 to 10 ng/ml and 0 to 300 ng/ml. The soluble peptide was HLA-A*24:02WT-01(mu)CYTWNQMNL. The amino acid sequence of the peptide (CYTWNQMNL)' is listed as SEQ ID NO: 2 in the attached sequence listing. The immobilized anti-CD28 antibody is biotinylated against human CD28 mouse monoclonal antibody (selection CD28.2, BioLegend)

Japan (stock company) and magnetic beads (Dynabeads (trademark) M_28 〇 streptavidin, Life technology Japan Co., Ltd.) were incubated at room temperature for 30 minutes, and combined with biotin-avidin reaction. The 201245224 anti-CD28 antibody conjugated to the aforementioned magnetic beads is hereinafter referred to as a complex of biotinylated cd28 antibody and beads, or a solid phase anti-CD28 antibody. (3) Purification and proliferation of dendritic cells derived from autologous cord blood. Cord blood (IE) obtained from the Institute of Physical Chemistry: HCB〇747, HLA-A locus compatible: 24:02 And 〇 2: 〇 6) 25mL to 37. (: warm water bath solution; east 'move suspended in PBS added with a final concentration of 5% DEXTSERAN 4 (Terum〇 Japan) and a final concentration of 2.5% human serum albumin (Japan Red Cross). The white blood cells will be obtained The PBS supplemented with 5 mM EDTA and 2% human serum albumin was washed 3 times. Then, Dynal (trademark) immunomagnetic beads CD14 (Life technology japan) was added to each of the 108 cells in the aforementioned white blood cell suspension. The company was incubated at room temperature for 2 minutes or more. The CD 14-positive cells were separated from the single-sphere macrophages of the bead-loving beads by a magnetic particle separator (DynaMag-15, Lifetechnology japan). The fraction of the negative cells is as follows (4) for the purification and proliferation of CD3/CD28 positive cells. The isolated cDi4-positive mononuclear spheres are suspended at a concentration of 106/mL in 5% human AB-type serum. After moving to a 35mm culture dish (EZ-BindShut (trademark), Asahi Glass Co., Ltd.) in X-VIV0 (trademark) 15 medium, 50 ng/mL recombinant human granule macrophage colony stimulating factor (GM_CSF, Peprotech) with a weight of 50ng/mL Human IL-4 (IL_4, Pepr〇tech) was cultured. On the 6th day of culture, 50 ng/mL of prostaglandin E2 (manufactured by First Fine Chemical Co., Ltd.) and Picibanil at a final concentration of 1 〇# g/mL were added. (0K432, Roche Chinese and Foreign Pharmaceuticals). Further cultured for 1 day to obtain mature dendritic cells. 201245224 (4) Purification and proliferation of CD3/CD28 positive cells from cord blood for CD14 negative cell fraction isolated from section (3) Further, CD-positive cells were removed, and 25 μL of Dynal (trademark) immunomagnetic beads CD4 (Li fetechnology Japan Co., Ltd.) was added per 2×1 07 cells, and the mixture was mixed at room temperature 20° C. for 10 minutes or more. Magnetic separation was used. (DynaMag-i 5) After removing CD4 positive cells, 're-addition for each 1 〇 7 mononuclear spheres

Dynabeads (trademark), Teel 1 Expander CD3/CD28 (trademark, Lifetechnology Japan) 25yL' after 30 minutes of mixing on ice or at 4 ° C 'using magnetic particle separator (j) ynaMag-15)

CD3/CD28 positive cells were concentrated. The aforementioned CD28-positive cells were cultured in suspension at a concentration of 1〇6/mL in X-VIV0(Trade Mark) 15 medium supplemented with 5% human AB type serum and 300 ng/mL recombinant human IL-15 (Peprotech). The medium to which the cytokine was added was replaced every 2 to 3 days after the start of the culture. At this time, the number of viable cells was determined using Trypan Blue staining, and fresh medium to which 300 ng/mL of IL-15 was added was added so that the cell concentration was 10 cells/mL. At the stage where the total number of cells of the aforementioned CD3/CD28-positive cells reached 1 x 10, stimulation was carried out with soluble peptide and anti-CD28 immunobeads according to the procedure described below. When the proliferation of CD3/CD28-positive cells was fast, it was cultured in the 丄_νιν〇 (trademark) 15 medium supplemented with 5% human AB type serum and 1 ng/mL recombinant human U 7 (Peprotech) during the initial sputum period. On day 7 later, IL-7 was replaced with 300 ng/mL recombinant human IL_15 (pepr〇tech), and the same result was obtained in the shortest period of 19 days. (5) Induction with soluble wT-i peptide and solid-phase anti-cD28 antibody 15 201245224

WT-1 peptide specific CTL

When the total number of cells of CD3/CD28-positive cells reached ιχι〇6, the first stimulation was performed with the immobilized anti-CD28 antibody and the soluble peptide, and the second stimulation was performed on the 7th day after the first stimulation. X-Vi v〇(Trade Mark) 15 medium supplemented with 5% human AB type jk clear and 30 0ng/mL recombinant human IL-1 5 was added with a final concentration of 1 〇V g/mL of soluble peptide HLA- A*24: 02 WT-l (niU) CYTWNQMNL and a medium for immobilizing anti-CD28 antibody for CTL induction. At this time, the bead concentration was adjusted so that the ratio of the number of magnetic immunobeads to T cells was 4: 1, and after the medium was changed, the beads were simultaneously diluted with the cells. (6) Proliferation after WT-1 peptide specific CTL induction The medium after CTL induction was replaced with a soluble peptide with a final concentration of 10 " g/mL, 5% human AB serum and 3〇 〇ng/recombinant human 11^-15 χ-νινο (trademark) 15 medium. It takes 19 days to increase the CTL, and it takes 4 days to grow.

(7) Using the same HLA-like terminal blood-derived dendritic cells to induce WT-1 peptide specific CTL (7.1) blood collection from healthy 2 self-suppressed subjects (HLA_A locus) Compatible: HLA-A*24:02 and 02:07, HLA_A*24:〇2 and 26:〇1)

A little blood. This laboratory is accredited by the Ethics Review Committee of the Institute of Medical Sciences of the University of Tokyo (Approved No. 20-56~〇21〇, Initial Recognition Date: February 10, 2011), Change Approval Date: Heisei 22 ij month i 9 After the implementation of the day, and the above-mentioned voluntary subjects agreed in writing. The blood collection system used a vacuum blood collection tube (TERUMO VENOJECT II EDTA-2Na) equipped with a 2i~22G 16 201245224 needle. (7.2) Prepare the dendritic cells from the terminal blood to dilute the blood obtained by keeping the diluted solution (pBS) at room temperature twice, and dilute the blood in each centrifuge tube layer by 2〇35mL and Fic〇n paque (specific gravity). 1.077' GE Healthcare Japan (shares company) 1〇~i5mL. Centrifuge at 50 0xg for 3 minutes at room temperature and stop without applying a brake. The centripetal supernatant (plasma fraction) was retained in a few ml and removed, and the intermediate layer was recovered. The foregoing intermediate layer recovered from 1 to 2 centrifuge tubes was collected into a new centrifuge tube of the roots, and a volume of 50 mL was prepared from the above dilution. The second centrifugation was carried out at 5 〇〇 x g, room temperature, and 1 minute. The supernatant was removed, and the pellet was suspended in 3 mL of the aforementioned dilution. The third centrifugation was carried out at 500 ng, room temperature, and 5 minutes. The supernatant was removed, and the pellet was suspended in PBS supplemented with 2 mM EDTA and 1% human blood/moon albumin to make the cell concentration 7 (hereinafter referred to as "mononuclear sphere suspension"). In the aforementioned mononuclear ball suspension, Dynal (trademark) immunomagnetic beads CD14 (Ufe techn〇i〇a) were added for each of the 8 mononuclear spheres.

Japan (stock) company 25/z L, stirred at 4t for 3 minutes. After the reaction, CD14-positive mononuclear spheres were separated using a magnetic particle separator (DynaMag_15). The nucleus nucleus was cultured in x-VIV(R) (trademark) 15 medium supplemented with 50 ng/mL of GM_CSF, 5 ng/mL of 1 b 4, 5% human AB type serum. The medium was not changed during the 6-day culture period. On the sixth day of culture, 5Gn (10) of prostaglandin E2 and ι〇μ g/mL of Picibanil were added and cultured for another day to obtain mature dendritic cells.

(7·3) CTL using a dendritic cell derived from human peripheral blood to induce a specific peptide CTL 17 201245224 The phosphine-derived CD3/CD28 positive cells purified and proliferated according to the procedure of Section (4) are added. In human x-mo (trademark) 15 medium containing 5% human Αβ-type serum and 3 〇〇η_, it was cultured under the conditions of 3rc and 5% c〇2. The CTL induction was performed for the first time in which the total number of cells reached ΐχΐ〇6, and the second stimulation on the 7th day after the first stimulation was performed twice. In the culture medium of the mature dendritic cells of the blood source prepared by the procedure described in (7.2), the addition of the soluble peptide HU-A*24: 〇2WT-1 (mu) CYTWNQMNL (HLA_A*24: 〇2 donor) or hla-A*02:01wt-i RMFPNAPYL (HLA_A*〇2: 〇1 when donated), stir at 37 ° C for 3 hours to make the above peptide Binding to the HLA class I molecule ± on the aforementioned dendritic cells. [The mature dendritic cells were mixed with the aforementioned (10) positive cells in a ratio of 1:5 to 1:6.7 in a sputum 2 well plate, and then co-cultured for 12 days after the first stimulation. (8) Verification of the reliability of the fluorescent-labeled tetravalent test substance In order to determine the proportion of the cells in the induced cytotoxic tau cells that recognize the 丨-丨 peptide antigen, the solid phase on the HLA_A*24: 〇2 molecule was used. Four beads of WT-1 peptide consisting of a large variant amino acid sequence and fluorescently labeled with pE (hereinafter referred to as "PE labeled HLA-A*24: 02WT-1 (mu)-quaternary body" However, the bead has been bound by a covalent bond, and the biotinylated PE labeled quaternary tetrapeptide of WT-1 peptide refers to the combination of specific interaction of avidin-biotin. The anti-CD28 antibody used for induction is also biotinylated' and immobilized to the avidin-binding beads. However, the immobilized anti-28 antibody is used, if it remains in the CTL for detecting the specificity of the ψτ-1 peptide. A sample of the flow cytometer, pE-labeled 18 201245224 ΙίίΑ-Α*24:02νΤ-1(ιηυ)- part of the quaternary bead may have a biotinylated ΡΕ-labeled WT-1 peptide The quaternary body falls off and replaces the biotinylated anti-CD28 antibody. If this shedding or substitution occurs, when the test is successful, In the case of specific CTL, the cells expressing CD28 also responded. Therefore, the following experiments were used to verify the possibility of shedding and substitution. From the HLA-A*24:02 positive cord blood 2 sample, according to (4) The procedure described in the section obtains CD3/CD28-positive cells. The aforementioned cells are supplemented with X-VIV0 (1,5 IU/mL of IL-2, 300 ng/mL of IL-1 5 and 5% of human AB-type serum). Trademark) 15 culture for 42 days. In addition, CD3/CD28 positive cells were isolated from HLA-A*020 1 positive healthy volunteers according to the procedure described in Section 7.3, with the addition of 300 IU/mL of IL-2. 30-ng/mL IL-15, 5% human AB-type Jinqing's χ-vivo(trademark) 15 was cultured for 21 days. For each 〇6 CD3/CD28 positive from the aforementioned human umbilical cord blood or healthy normal human peripheral blood The cells were supplemented with biotinylated anti-human CD28 mouse monoclonal antibody (CD28, BioLegend Japan Co., Ltd.) 20//L, at 0 to 4. (: Stir for 30 minutes. Thereafter, add PE-labeled HLA-A *24 : 02WT-1 (mu)CYTWNQMNL quaternary body (when HLA-A*24:02 is donated), or PE-labeled HLA-A wood 02:01WT-1 RMFPNAPYL quaternary body (HLA-A hibiscus 2 :〇1 Donate

When incubated at room temperature for 20 minutes, the quaternary body was bound to the tau cell receptor of the aforementioned cells. Then, a secondary antibody for binding to the cell surface of the anti-human CD28 mouse monoclonal antibody was added, that is, APC-labeled anti-mouse j gG scorpion monoclonal antibody (eBioecience) 2 〇al, at 0 to 4 ° C Stir for 20 minutes' to verify the binding of biotinylated CD28 antibody on the cells by flow cytometry. 19 201245224

(9) CTL for detecting WT-1 peptide specificity The first stimulation of the WT_丨-peptide specific CTL can be performed in any of the above three procedures at a stage of reaching a total number of cells of 1×1 〇6, and The second stimulation of the second day after the first stimulation, the umbilical cord blood-derived CD3/CD28-positive cells, which were stimulated twice, were recovered on the 12th day of the first stimulation, and the anti-human CD3 monoclonal mouse IgG2a antibody was labeled with F iTC (selection Body HlT3a

BioLegend Japan Co., Ltd., APC-labeled anti-CD8 antibody, pE-labeled HLA-A*24:02WT-l(mu)-quaternary body were subjected to triple staining and analyzed by flow cytometry. 2. Results FIGS. 2A to 2H show the first stimulation for the stage of the total number of cells up to 1×10 in the above three procedures, and the second stimulation of the second stimulation on the 7th day after the first stimulation. Cord-derived CD8-positive cells treated with peptide-specific CTL were specifically identified in cells of CD3+ gates on day 12 after initial stimulation, specifically identifying HLA-A*24: 〇2WT_1 (mu cells of quaternary cells) (The following is a two-dimensional flow cytometer analysis result of "quaternary body positive cells". The vertical axis of the results of FIGS. 2A to 2 is the fluorescence intensity of the pE mark, and the horizontal axis represents the fluorescence intensity of the APC mark. In the samples of FIGS. 2A and B, the above three kinds of induction treatments were not performed, and only χ_νιν〇 (trademark Μ5 medium) with the addition of (10) human AB type serum and 300 ng/mL recombinant human IL_15 was replaced with FIG. 2C and The experiment of Figure 2D cultured CD8-positive cells of the same day number of blood-derived blood. The samples of Figures 2C and 2 were firstly stimulated at a stage of 1 x 16 cells, and 7 days after the first stimulation. The second stimulation used a total of 2 times to use soluble (5) wins and anti-20 201245224 .CD After the induction of WT-1 peptide-specific cTL by 28 immunoglobulins, the cd8-positive cells derived from cord blood of the second day after the first stimulation were cultured. The samples of FIG. 2E and F were applied to the total cells. The first stimulation at the stage of 1χ1〇6, and the 2nd stimulation on the 7th day after the first stimulation, the WT-i peptide was added to the dendritic cells derived from the same terminal blood using the WT-1 mutant peptide. After the induction treatment of specific CTL, the cord blood-derived CD8-positive cells were cultured until the 12th day after the first stimulation. The samples of Fig. 2G and sputum were firstly stimulated at the stage of total cell number lxl〇e And the second stimulation on the 7th day after the first stimulation, the WT-1 peptide specific CTL induction treatment was carried out by using the mutant peptide to add autologous cord blood mononuclear-derived dendritic cells. Cord-derived CD8-positive cells up to day 12 after the first stimulation. Samples of the results of Figures 2A, C, E, and G were mixed with FITC-labeled anti-C])3 antibody and standard s-anti-CD8 antibody And staining. Samples of the results of Figures 2B, d, F, and η, with FITC markers CD3 antibody, mixed with APC-labeled anti-CD8 antibody, ΡΕ-labeled HLA-A*24: 02WT-1 (mu)-quaternary body and stained. As shown in Figure 2D, using soluble conjugated peptide and anti-CD28 immunobeads Among the cord blood-derived CD3/CD28-positive cells induced by WT-1 peptide-specific CTL, about 8.2% of cells passing CD+3 gates were tetramer-positive 'that is, 'specifically identified as HLA -A*24: 02 The WT-1 source peptide of the dual gene and is the cancer antigen. WT-1 specific cytotoxic tau cells will overexpress WT-1 malignant cell responses with lymphoma, but it is known that it does not attack normal cells that exhibit only a small amount of WT-1 (Gao, L. et al. ,

Blood, 95:2 1 98-2203, (2000), Oka, Y. et al., Curr. 〇p 21 201245224 in Immunol. 20:211-220 (2008)). In contrast, as shown in FIG. 2F, the WT-1 mutant peptide was added to the dendritic cells derived from the terminal blood, and the WT-1 peptide-specific CTL-induced cord blood-derived CD8-positive cells were passed. The CD+3 gate cells are only about 〇. 22% are specifically identified as the HLA-A* 2 4 : 0 2 dual gene 脉络 and the cancer antigen is the big moon HLA-A*24:02WT-l(mu) CYTWNQMNL. Further, as shown in Fig. 2H, WT-1 mutant peptide was added to autologous umbilical cord blood mononuclear-derived dendritic cells to carry out WT-1 peptide-specific CTL-induced cord blood-derived CD3 positive cells, through CD + The cells of the 3 gates are only about 〇. 55% are specifically identified as the domain of the HLA-A*24:02 dual gene and the peptide of the cancer antigen HLA-A*24:02WT-l(nui)CYTWNQMNL. Further, as shown in Fig. 2B, cord blood-derived CD8-positive cells which were not subjected to the control experiment of WT-1 peptide-specific CTL induction, only about 28% of the cells passing through the CD + 3 gate were specifically identified as HLA- A*24: 02 is the vein of the dual gene and is the peptide of the cancer antigen HLA-A*24: 02 WT-l (mu) CYTWNQMNL. As shown in Figures 2A, C, E, and G, 'samples stained with unlabeled PE-labeled HLA-Am:G2WT-1(mu)-quaternary body and only mixed with APC-labeled anti-(10) antibody, passed through (10) + closed cells Among them, the cells showing fluorescence at the emission wavelength of PE are 〇. (10). As described above, 'from the results of Figs. 2A to H', the above-mentioned three kinds of treatments with the specificity of the singularity ctl, 'utilizing the soluble WT' peptide and the anti-CD28 immunobead

The implementation of the WT-1 peptide specificity Γττ 铦 老 抑 诱导 诱导 远 远 远 远 远 远 远 远 远 远 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识 辨识And the results of the experiment of '(8), Tim, 』, plus or without biotinylated CD28 antibody did not appear quaternary positive abortion ·, ^ can be confirmed quaternary body using avidin _ 22 201245224 The possibility that the white pigment binds to become a false positive is extremely low. Example 2 CTL which induces WT-1 derivative WT-1 peptide specificity from cord blood (2) 1. Materials and methods Cord blood different from Example 1 (ID) No.: HCB00751, compatible type of HLA-1 locus 24.no n ▲ 02 and 33: 03) CD8-positive cells derived from the same type, and the same kind of induction treatment as that of Example 1 was given. Flow cytometry analysis of polymer positive cells. 2. Results Figure 3 shows no effect on the first stimulation of the 6-day phase of the 7th day after the first stimulation, with the soluble peptide and the anti-28 immunobead Two-dimensional flow of quaternary positive cells in umbilical cord blood-derived (10) positive cells stimulated by a total of two WT-1 peptide-specific CTLs in the CD3 gated cells on the 12th day after the initial stimulation The analysis result of the fine counter. The vertical axis of the result graph of Fig. 3 is the fluorescence intensity of the pE mark, and the plate axis is the fluorescence intensity of the APC mark. The sample of Figure 3 is the first stimulation of the total cell & G6 stage, and the second stimulation of the 7th day after the first stimulation is performed twice with the soluble WT_i peptide and anti-28 immune beads. After inducing treatment of ψτ-1 peptide-specific ctl, the cord-derived CD8-positive cell line cultured until the first day after the first stimulation will be FITC-labeled anti-CD3 antibody, and APC-labeled anti-CD8 antibody, 5 hexyl 111^4*24:02\^-1 (11111) quaternary bodies were mixed and stained. As shown in Fig. 3, cord blood-derived cD8-positive cells different from Example i were also used for solubilization. - 丨 peptide and anti-CD28 immunoglobulin 23 201245224 WT 1 peptide-specific CTL-induced umbilical cord blood-derived cd3/CD28 positive cells, about 94% of CD+ cells are tetramer-positive In contrast, when the umbilical cord blood-derived dendritic cells are used for the same umbilical cord blood, the WT-i peptide-specific CTL induction is used, and the same HLA-derived blood-derived dendritic cells are used. When WT_i peptide specific CTL induction was performed, 36% of cells passed through the cd+ gate, 33. 33% is tetramer-positive (not shown). The umbilical cord blood-derived CD3/CD28-positive cells that have not been subjected to WT_i peptide specificity ctl-induced control have only about 0.52% specificity. Recognized as HLA_A*2^〇2 dual

The vein of the gene and the peptide of the source of the cancer antigen. Unmixed pE private 5}11^4*24:02 set-1 (1111〇-quaternary body, but 1?11' (: labeled anti-CD3 antibody, mixed with APC-labeled anti-CD8 antibody and stained sample Among the cells that passed through the CD3+ gate, the fluorescence wavelength of the PE showed that the fluorescence of the cells was 0·0.5% or less. From the above results, in the experiment using most cord blood, the soluble WT-1 peptide and the anti-CD28 were used for immunization. The CTL induction treatment of the wt-1 peptide specificity performed by the beads was far more than that of the other two induction treatments, indicating that more CTLs for identifying the WT-1 peptide were induced. It has been reported that the clinical treatment of cancer treatment using the peptide vaccine is reported. The ratio of quaternary positive cells in the case of vaccine sensation in the patient during the test. For example, according to 〇ka γ et al. (Pr〇c Natl Acad)

Sci USA, 1 01:1 3885 (2004) reported that when there were 2 breast cancer patients, 10 lung cancer patients, and 14 leukemia patients, the wild-type or subtype HLA-A*24:02 restricted WT- 1 peptide (〇.3mg_30mg) and Freud's incomplete adjuvant were used to perform three times of feeling at 2 week intervals, and felt as WT-1 quaternary positive CD8 land κ+ λ & The ratio of + 1 positive cells is the central value of 0. The training range is mm), and the ratio of WT1 quaternary positive CD8 positive cells after the induction is the central value 〇 34% (range: 〇ιι_6·6ι%). In addition, according to Van Tende 1 〇〇VF 箄人 ru KI , · , „ 寻 ( (pr〇c Natl Aead Sei USl 107:13824 (2010)) report, for the warp a to affixed with & Acute bone marrow white blood

In patients with disease (AML), subcutaneous inoculation of autologous dendritic cells into which mRNA genes of WT ^ and negative ww proteins were introduced, Quaternary positive cells detected by Shishidu ^ a , ^ τ in the last blood Less than 1%. No report was found on the proportion of quaternary positive cells that were shown to be positive in the test tube using the peptide antigen restricted by _ Di Ι | μ. Example 3

Single-induction of CTL from cord blood 1. Materials and methods According to the procedure described in Example 1, different cord blood was confirmed to be hla_a*24:〇2 positive (ID number: hcb〇〇756, hla locus) Phase-type: 24:02 and 33:03) separation. 014 negative 〇) 4 negative 〇 03/0028 positive cells). The cells were proliferated at 37 ° C, 5% CO 2 at the beginning of the week with x_VIV(R) (15) medium supplemented with 1 ng/mL of IL_7 and 5% human AB type serum, and then every 2 to 3 X-VIV0 (trademark) i5 medium supplemented with 3 ng/mL of IL-15 and 5% human AB type serum was added to the day, and the optimum cell concentration was 1 〇 6 cells/mL, and proliferation was continued for 28 days. Thereafter, the above-mentioned cells were suspended in Cellbanker (trademark) (Xi Ci to "share" company) and stored frozen in liquid nitrogen, and thawed at the time of use for the following experiment. 5% human AB type serum was added, 3 添加The ng/mL IL-15 X-VIV0 (Business 25 201245224 standard) 15 medium was further supplemented with biotinylated CD28 antibody and bead complex, cancer antigen WT-1 specific variation soluble peptide HLA-A*24: Thawed CD3/CD28 positive cells were cultured for 1 day in the medium of 02WT-l(mu)CYTWNQMNL or CMVpp65 specific peptide HLA-A*24:02CMVpp65 QYDPVAALF (SEQ ID NO: 5). The cells were recovered in PBS, and for each 〇6 cells, a PE-labeled HLA-A*24:02WT-l(mu)CYTWNQMNL quaternary body, or a PE-labeled HLA-A*24:02CMV pp65 QYDPVAALF quaternary body was added. After standing at room temperature for 20 minutes, 2 〇m 1 of FITC-labeled anti-CD3 monoclonal mouse IgG2a antibody (selected by HIT3a, manufactured by Biolegend JAPAN Co., Ltd.) and APC-labeled anti-CD8 antibody were added. The colony RPA-T8, BioLegend JAPAN (shares) company, after stirring at 0~4 °C for 20 minutes, the flow is fine Analysis by cell counter. 2. Results The results of analysis by the same flow cytometer as in Examples 1 and 2 were based on the number of CD8-positive cells derived from cord blood that had been frozen after the initial culture, and then re-thawed to prepare the total number of cells. Lxl〇6 stages with the addition of the cancer antigen WT-1 specific variation soluble peptide HLA-A*24:02WT-l(mu)CYTWNQMNL, or CMVpp65 specific soluble peptide HLA-A*24:02QYDPVAALF, After stimulation with the medium of CD28 immunobeads, the cells were cultured to the first day after stimulation, and in the cells passing through the 匸03+ gate, the #1'-1 peptide quaternary positive cells were about 2.72% ( In the same manner, the cells were added to the medium supplemented with CMVpp65-specific soluble peptide HLA-A*24:02QYDPVAALF and CD28 immunobeads, and then cultured until the 10th day after stimulation, and the cells were passed. In the cells of the CD3+ gate, the CMVpp65 peptide quaternary tough cells were about 3.99% (not shown). The cord blood-derived CD3 positive cells of the control experiment without the peptide specific CTL induction were labeled with PE. -1 peptide quaternary body mixed with pE-labeled CMVpp65 peptide tetramer The stained sample, after one stimulation, was cultured to the first day after stimulation. The cells in the cells passing through the CD3+ gate, through the cells of the CD 3 gate, showed a 0.77% of the cells at the abundance wavelength of PE. (not shown). Cord-derived CD3-positive cells induced by WT-1 1-specific CTL were not mixed with PE-labeled WT-1 peptide tetramers and only stained with APC-labeled anti-CD8 antibody, and passed through CD3 + gates. The cells in the cells showed fluorescence at the fluorescence wavelength of PE of 0.19% (not shown). There were cord blood-derived CD3-positive cells induced by CMVpp65 peptide-specific CTL, and samples stained with only the APC-labeled anti-CD8 antibody mixed with the PE-labeled CMVpp65 peptide tetramer, in the cells passing through the CD3+ gate The cells showing fluorescence at the fluorescence wavelength of pe were 0 · 2 7% (not shown). Cord-derived CD3-positive cells without a peptide-specific CTL-induced control experiment, without pe-labeled WT-1 peptide quaternary or PE-labeled CMVpp65 peptide quaternary and only mixed with APC-labeled anti-CD8 antibody The stained cells showed a fluorescent cell at a fluorescence wavelength of pE of 0.14% (not shown). From the results of the present example, the treatment steps of WT-1 peptide specific CTL induction using the soluble WT-1 peptide and the anti-CD28 immunobead were shown to have an effect only once. Further, peptides using soluble peptides and anti-CD28 immunobeads were specifically induced by CTLs, which were not limited to WT-1 peptides, and were also effective in CMVpp65 peptides. The peptide-specific CTL induction method of the present invention in which a soluble peptide and an anti-CD28 immunobead are used in combination, regardless of the type of the peptide antigen, can be implemented. This system determines the amino acid sequence of the peptide that acts as an antigen in response to the HLA-compatible form of the patient, and can induce and treat most cytotoxic T(4) regardless of tumor antigen or viral antigen. Further, by using a soluble peptide as a peptide antigen, the antigen can be easily changed even if the tumor antigen and the virus are mutated to obtain a vaccine-tolerant escape mutation. Further, in the present example, even if the CTL induction of i times is effective, it can be expected to be carried out by a simpler culture implementation step than the conventional CTL induction method. Example 4 A slight blood-induced WT-1 peptide specificity ctl 1. Materials and methods k are the same healthy volunteer subjects as in Example 1 (7.1) (HLA-a locus compatible type) :HLA-A*24:〇2& 26:〇1) Take the last blood. The blood was collected in the same procedure as in Example 1 (7.1), and a mononuclear ball suspension was prepared in the same procedure as in Example 1 (7.2), and the CD"positive mononuclear sphere was removed from the aforementioned mononuclear ball suspension, from The remaining CD14 negative fractions were used to refine CD8 positive cells using the group (Lifetechnologies japan). Specifically, the biotinylated anti-CD8 antibody is reacted with the human terminal blood mononuclear ball suspension CD14 negative fraction on ice (ο~) for 1 minute, and then bound to the avidin on the magnetic immunobead on ice (〇 ~4t) Reaction 15 minutes The CD8 positive cells were isolated using a magnetic particle separator (DynaMag-15). Thereafter, a release buffer was added at room temperature and incubated for 1 minute to allow the aforementioned avidin to bind to the magnetic immunoglobulin to be released from the CD8 positive lymphocytes. The recovered CD8-positive fine 28 201245224 cells were cultured in X-VIV0 (Trade Mark) 15 supplemented with 5% human AB-type serum and 300 ng/mL IL-15 at 37 ° C under 5% C 2 . After the total cell number was prepared as lxl 06, the cancer antigen WT-1 specific mutation soluble peptide HLA-A*24:02WT-l(mu)CYTWNQMNL, or CMVpp65 specific peptide hlA-A*24 was added. The medium of 02QYDPVAALF and CD28 immunobeads was stimulated for the first time, and after the same stimulation on the 7th day after the first stimulation, the culture was continued until the 11th day after the first stimulation. Every 2 to 3 days, diluted with fresh medium supplemented with IL-15 to make the cell concentration ι〇6 L. On the 11th day after human stimulation, the quaternary positive rate was measured by flow cytometry. Analysis. 2. Results and Examples 3 The results of the same flow cytometry analysis were carried out for the addition of the cancer antigen WT-1 specific variant soluble peptide HLA-A*24:02WT-l(mu)CYTMNQMNL and CD28 The medium of the immunobeads was first stimulated at a stage where the total number of cells reached lxl〇6, and after the same stimulation on the 7th day after the first stimulation, the culture was continued until the end of the first stimulation, the peptide specific CTL induction treatment on the uth day after the first stimulation. The WT-1 peptide tetra 70 cation II cells were about 5.33% (not shown), and the cells of the CD8+ cells were incubated on the 11th day after the start of the culture. In the control experiment, the terminal blood-derived (10) positive cells were not subjected to the CTL-induced CTL control. In the first culture of the first culture, "n Q + Ρθ ΛΑ Α αΑ was passed through the CD3 gate cells, WT- About 0.15% of i-peptide positive cells (not shown). The same from the peripheral blood of the sex cells, using (10) secret soluble peptide and anti-cafe 8 immune L nutrition start f and the 7th day of the 2 stimulation At the time of the 11th 29th, 2012, 24, 24th day of the culture, the cells of the CD3+ passed through the cells of the CD3+, and the wt] peptide quaternary positive cells were about 4.53% (not shown). The FITC marker was mixed without mixing the PE labeled wry peptide quaternary body. The anti-CD3 antibody and the APC-labeled anti-CD8 antibody stained the sample, and the cells showing fluorescence at the fluorescence wavelength of PE were 0·13% (not shown) among the cells passing through the CD3+ gate. Example 5 From the umbilical cord Blood-induced peptide specificity of human telomerase hTERT

CTL 1. Materials and methods from different cord blood confirmed to be HLA-A*24: 02 positive (ID number: HCB01100, compatible type of HLA-A locus: 24:02 and 26·〇1), according to The procedure described in Example 1 (3) was used to isolate cj using magnetic immunobeads. 14 Negative CD4-negative CD3/CD28 positive cells. The above cells were diluted to a concentration of 1 〇 6 / mL in X-VIV0 (Trade Mark) 15 medium supplemented with 10 ng/mL of IL-7 and 5% human AB type serum 'at 37 ° C, 5% C The 〇2 concentration was proliferated during the first 1 week of the start of culture, and then from the 7th day, X-VIVO (trademark) 15 medium supplemented with 300 ng/mL of il-15 and 5% human AB type serum was added every 2~ Each dilution was performed once every 3 days to obtain an optimum cell concentration of 1 / mL. At the stage where the total number of cells reached lxlO6, the first stimulation was performed with the medium supplemented with hTERT-derived soluble peptide HLA-A*24:02 VYGFVRACL (SEQ ID NO: 3) and CD28 immunobeads, and then on the 7th day after the first stimulation. The same stimulation was performed 'after incubation until the 14th day after the first stimulation. At the time of stimulation, the complex of the imprinted CD28 antibody and the bead was added at a bead:cell ratio of 4:1 at 30 201245224. The cells were recovered in PBS on the 14th day after the first stimulation, and 20# L of ΡΕ-labeled HLA-A*24:0 2hTERT VYGFVRACL quaternary body was added every 106 cells for 20 minutes at room temperature, and 20#L was added. FITC-labeled anti-human CD3 monoclonal mouse igG2a antibody (selected HIT3a, BioLegend Japan Co., Ltd.) and APC-labeled anti-CD8 antibody (selected RPA-T8, BioLegend Japan), at 0~ Stir at 4 ° C for 20 minutes and analyze by flow cytometry. 2. Results Figures 4A to D are the results of two-dimensional flow cytometry analysis using FITC-labeled anti-CD3 antibody and PE-labeled HLA-A*24:02hTERT VYGFVRACL quaternary and APC-labeled human anti-CD8 antibody. The vertical axis of the graph of Figs. 4A to 4D is the fluorescence intensity of the PE mark, and the horizontal axis is the fluorescence intensity of the APC mark. For the addition of hTERT soluble peptide

HLA-A*24:02VYGFVRACL and CD28 immunobead culture medium The hTERT-derived CD8-positive cells were induced by hTERT-derived peptide-specific CTL induction, and hTERT peptides were passed through CD3+ gate cells on the 14th day after the first stimulation. The quaternary positive cells were approximately 74.7% (Fig. 4C). The umbilical cord blood-derived CD3 positive cells of the control experiment in which the peptide-specific CTL induction was not carried out showed hTERT peptide quaternary positive cells of about 0.06% among the cells that passed through the CD3+ gate on the 11th day of culture initiation (Fig. 4B). Cord blood-derived CD8 1% cells do not mix PE-labeled hTERT peptide with hTERT-derived peptide-specific CTL induction treatment with medium supplemented with hTERT soluble peptide HLA-A*24:02 VYGFVRACL and CD28 immunobeads 8%的图(图图) The cells in the luminescence of the luminescence of the cells are 0. 08% or less in the cells of the CD3+ gate. 4D). Umbilical blood-derived CD3-positive cells that did not undergo peptide-specific CTL-induced control experiments were not mixed with pe-labeled hTERT-like quaternary bodies and stained with FITC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody. Among the cells passing through the CD3+ gate, the fluorescent cells at the fluorescence wavelength of PE showed 〇. 〇 2% (Fig. 4A). Example 6 Inducing survivin_2B specificity from cord blood to peptide specificity ctl 1. Materials and methods From different cord bloods that have been confirmed to be HLA-A*24: 02 positive (id number: HCB01100, HLA-A locus Compatible: 24:02 and 26:01), CD丄4 negative CD4 negative CD3/CD28 positive cells were isolated using magnetic immunobeads according to the procedure described in Example 1 (3). The above cells were diluted to a concentration of 1 〇 6 / mL in the X-VIVO (trademark) 15 medium supplemented with 1 ng/mL of IL-7 and 5% of human AB type sera. (:, 5% C〇2 concentration was proliferated in the first 1 week of the start of culture, and from the 7th day, X-VIVO with 3〇Ong/mL of IL-15 and 5% of human AB type serum was added ( The trademark 15 medium was diluted once every 2 to 3 days to obtain an optimum cell concentration of 1 〇 6 cells/mL. The total cell number reached 1x10, and the survivin-2B-derived soluble peptide HLA-A* was added. The medium of 24:02 survivin-2B AYACNTSTL (SEQ ID NO: 4) and CD28 immunobeads was stimulated for the first time, and then the same stimulation was performed on the 7th day after the first stimulation, and then cultured until the first day after the first stimulation. The complex of biotinylated CD28 antibody and beads was added at a concentration of 4:1 of the bead:cell ratio 32 201245224. The cells were recovered as pBS on the 1st day after the first stimulation, and 2 〇eL of PE was added per 106 cells. Labeled HLA-A*24:02 survivin-2B AYACNTSTL quaternary body, after standing at room temperature for 20 minutes, add 20# L of FITC-labeled anti-human CD3 monoclonal mouse IgG2a antibody (selection HIT3a, BioLegend Japan) (share) company) with APC-labeled anti-CD8 antibody (choose lean body rpa-T8, BioLegend Japan) 〇 〇 ~ 4 C stir for 20 minutes 'flow analysis by flow cytometer. 2. Results Figure 5A ~ D for the use of FITC-labeled anti-CD3 antibody and PE labeled HLA-A*24:02 survivin-2B AYACNTSTL· quaternary body The results of two-dimensional flow cytometry obtained from PE and APC in cells stained with APC-labeled human anti-CD8 antibody and passed through CD3+ gate. The vertical axis of the results of Figures 5a-d is PE-labeled fluorescence. Intensity 'the horizontal axis is the fluorescence of the apc mark

strength. For the first stimulation with the addition of HLA-A*24: 02 survivin-2B AYACNTSTL soluble peptide and anti-CD28 immunobeads to the total number of cells up to 1 χ丨〇6, and then 7th after the first stimulation After the same stimulation, the cord blood-derived CD8-positive cells cultured on the first day after the first stimulation were stained with FITC-labeled anti-CD3 antibody and pe-labeled HLA-A*24: 02hTERT quaternary and APC-labeled anti-CD8 antibody. Among the cells that passed the CD3+ gate, the SUrvivin-2B peptide quaternary positive cells were about 116% (Fig. 5C). A sample stained with a pe-labeled survivin_2B peptide quaternary body and labeled with an anti-CD3 antibody and an APC-labeled anti-CD8 antibody, in the cells passing through the (10) gate, the force PE @fluorescence wavelength showed a fluorescent cell of 0.000. Below % (Figure 5D). The umbilical cord blood-derived CD3-positive cells of the control-independent CTL-induced control were not tested. In the cells that passed the CD3+ gate on the first day of culture, the survivin_2B peptide quaternary positive cells were approximately 〇. 03 % (Fig. 5B). Samples stained with FISC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody were mixed with PE-labeled survivin_2B peptide quaternary body. In the cells passing CD3+ gate, the fluorescence wavelength of pE was shown to be 〇. 03% (Fig. 5A). Example 7 Simultaneous induction of CTLs specific for four peptides from cord blood 1. Materials and methods Different cord bloods that have been confirmed to be HLA-A*24:02 positive (ID compilation: RC2R1 0010, HLA- Compatible type of A locus: 24: 〇 2 / blank may be 24 : 02 homozygous) After removal of mononuclear spheres by attachment, CD4 negative CD8 positive cells were isolated using magnetic immunobeads according to the procedure described in the examples. . The cells were cultured at a concentration of 1〇6/mL with χ_νιν〇(trademark) 15 medium supplemented with 3〇〇ng/mL IL_i5 and 5% human AB type serum, and the total number of cells was lx106. The first stimulation was carried out with a medium supplemented with the following four peptides and CD28 immunobeads with HU-α*24:〇2 restriction. The peptides added were: (1) WT-1 specific variant peptide CYTWNQMNL, (2) CMVpp65 peptide QYDPVAALF, (3) hTERT specific peptide VYGFVRACL, and (4) survivin-2B specific peptide AYACNTSTL 4 kinds. After the same stimulation on the 7th day after the first stimulation, the culture was continued until the 12th day after the first stimulation. In the case of stimulation, in addition to the peptide, the complex of biotinylated CD28 antibody and beads was added at a concentration of beads of 4". The cells were diluted once every 2 to 3 days to become optimal cells 34 201245224 Concentration 106 cells/mL, and the state was proliferated to the first day of the first stimulation. Recover cells in PBS on day 12 'pE-labeled HLA-A*24:02 WT-l(mu)-quaternary or PE-labeled HLA-A*24:02CMVpp65 quaternary or each 1 〇6 cells PE label HLA-A*24:02 hTERT quaternary or PE label HLA-A*24:02 survivin-2B quaternary body 20// L ' After standing for 20 minutes at room temperature, add FITC-labeled anti-human CD3 Monoclonal mouse lgG2a antibody (selected HIT3a, BioLegend Japan Co., Ltd.) and APC-labeled anti-CD8 antibody (selected rpa-T8, BioLegend Japan Co., Ltd.) 20/z L, 〇~4. (: 20 minutes of incorporation, flow cytometer analysis. 2. Results Figure 6 A to D shows anti-c D 3 antibody labeled with FIT C and p E labeled HLA-A*24:02WT-l(mu)- The results of the two-dimensional flow cytometry obtained by pe and APC among the cells stained with quaternary and APC-labeled anti-CD8 antibodies and passed through CD3+ gates. The vertical axis of the results of the graphs of Figures 6A-D is pE-labeled Light intensity, the horizontal axis is the fluorescence intensity of APC labeling. The total cell number is prepared as lxlO6 and then 'first stimulation with WT-1 specific variant peptide CYTWNQMNL and anti-CD28 immunobead, and then 7th after the first stimulation. The same stimulation was carried out in the day, and the CD8-positive cells derived from cord blood derived from the first day of stimulation for the second day were cultured, and the FIK-labeled anti-cd3 antibody source, PE-labeled HLA-A*WT-tetragen and APC-labeled anti-cD8 antibody were used. For staining, cells with CD3+ gates had a peptide-specific quaternary positive cell of approximately 〇89.9% (Fig. 6Α). For the utilization of hu_a*24: 〇2 cmvpp65 peptide and anti-CD28 immunobeads and Figure 6a Experiment with the same day

35 S 201245224 The number and staining of CD8-positive cells derived from cord blood, on the 12th day after the first stimulation, passed through the [03+ gate cells of the '〇^|\^?65-specific quaternary positive cells. 66% (Fig. 6B). For cord blood-derived CD8-positive cells that were stained with the same number of days and stained with hTj; RT-derived peptide and anti-CD28 immunobeads in the same experiment as in Figure 6A, passed through the cells of C£)3+ gate on the jth day after the first stimulation. The hTERT-specific quaternary positive cells were approximately 95% (Fig. 6C). For cord blood-derived CD8-positive cells that were stained with survivin-2B-derived soluble peptides and anti-CD28 immunobeads in the same day and stained as in the experiment of Figure 6A, in the cells of CD3+ gates on day 127 after the first stimulation, survivin- The 2B-specific quaternary positive cells were about 1.19% (Fig. 6D). The peptide-specific CTL induction treatment was not performed, and the cord blood-derived CD8-positive cells of the same day and stained with the experiments of Figs. 6A to 6D were passed through the CD3+ gate cells on the 12th day after the first stimulation, WT-1. The quaternary positive cells with a specificity of singularity were approximately 58% (Fig. 6E) ^The PE-labeled anti-CD3 antibody was mixed with the APC-labeled anti-CD8 antibody and stained without mixing the PE-labeled peptide-quaternary body. The sample, through the cells of the CD3+ gate, shows that the fluorescent cells at the emission wavelength of PE are 〇. 〇 5% (Fig. gf). From the results of the examples 'induction of WT-1 peptide specificity using a soluble WT-1 peptide and anti-CD28 immunobeads, not limited to cord blood-derived CD8-positive cells, at least in the final blood-derived cd8-positive Cells are also not suitable. Similarly, WT-1 peptide-specific CTLs induced by soluble CMVpp65 peptide and anti-CD28 immunobeads were not limited to CD8-positive cells derived from cord blood, and at least the cd8-positive cells derived from the terminal blood were not applicable. Furthermore, 'not limited to WT_i or CMVpp65, this method also shows that 36 201245224 cannot use other extensive cancer antigens such as hTERT or survivin-2B. Further, the composition for inducing cytotoxic T cells of the present invention using a soluble peptide and an immobilized anti-CD28 antibody can be applied to various CD8-positive cells derived from various hematopoietic stem cells. Thus, the composition for inducing cytotoxic T cells of the present invention can also be applied to hematopoietic stem cells in vivo other than terminal blood, for example, tissue-derived hematopoietic stem cell-derived CD8-positive cells such as bone marrow lymph nodes, or Embryonic stem cells' Hematopoietic stem cell-derived CD8-positive cells differentiated from adult stem cells and artificial pluripotent stem (iPS) cells. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the hypothesis regarding the action of the composition for inducing cytotoxic T cells of the present invention. 2A shows sputum-derived CD8-positive cells cultured without CTL induction treatment, stained with isothiocyanate luciferin (FITC)-labeled anti-CD3 antibody and isophycocyanin (APC)-labeled anti-CD8 antibody, Further, the results of analysis by the two-dimensional flow cytometry of measuring the intensity of the fluorescence wavelength of PE and APC in the cells of the CD3 + gate. Figure 2B shows cord blood-derived CD8-positive cells cultured without CTL induction treatment, FITC-labeled anti-CD3 antibody, PE-labeled HLA-A*24:02 WT-l(mu)-quaternary body, and APC-labeled antibody The CD8 antibody was stained and analyzed by a two-dimensional flow cytometer that measures the intensity of the fluorescence wavelength of PE and APC in the cells of the CD3+ gate. Figure 2C shows cord blood-derived CD8 positivity 37 201245224 spleen cells cultured with WT-1 peptide specific CTL induction with soluble WT-1 peptide and anti-CD28 immunobeads. FITC-labeled anti-CD3 antibody, labeled with APC The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of PE and APC in the cells stained with anti-CD8 antibody and passed through CD3+ gates. Figure 2D shows umbilical cord blood-derived CD8 positive cells cultured with WT-1 peptide-specific ctl induction with soluble WT-1 peptide and anti-CD28 immunobeads. FITC-labeled anti-CD3 antibody, labeled with PE HLA = The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of PE and APC in cells stained with A*24 (mu)-quaternary body and APC-labeled anti-CD8 antibody and passed through CD3+ gates. Figure 2E shows that cord blood-derived CD8-positive cells cultured with WT_丨 peptide specific CTL after dendritic cell-specific CTL induction with the addition of wt-1 mutant peptides were labeled with FITC-labeled anti-CD3 antibody, The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of PE and APC were measured by staining with APC-labeled anti-CD8 antibody and passing through CD3+ gates. Figure 2F shows umbilical cord blood-derived CD8-positive cells cultured with WT peptide-specific CTL-induced dendritic cells derived from allogeneic blood cells supplemented with WT-1 mutant peptide, labeled with anti-CD3 antibody by FITC, and PE shows the intensity of the fluorescence wavelength of PE and APC in the cells of HLA-A*24: 〇2WT-l(mu)_tetramer, stained with Apc-labeled anti-CD8 antibody, and passed through CD3+ gates. The results of the two-dimensional flow cytometry analysis. Figure 2G shows that the spleen-derived CD8-positive cells cultured from the umbilical cord blood mononuclear cells of the umbilical cord blood mononuclear cells with the addition of 0 WT-1 t peptides are ... FITC-labeled anti-CD3 antibody, 38 201245224 stained with APC-labeled anti-CD8 antibody, and the results of two-dimensional flow cytometry analysis of the intensity of fluorescence wavelengths of PE and APC were measured among cells of CD3+ gate. Figure 2H shows umbilical cord blood-derived CD8-positive cells cultured against dendritic cells derived from autologous cord blood mononuclear cells supplemented with WT-1 mutant peptide by WT_丨 peptide specific CTL induction, with F丨TC Labeling anti-CD3 antibodies, with 标 标 § 111 ^ 4 * 24: 02 $ 1 '-1 (11111) - quaternary body and eight? (: The result of two-dimensional flow cytometry analysis in which the intensity of the fluorescence wavelength of pe and Apc was measured by staining with the anti-(:1)8 antibody and by the CD3+ gate. Figure 3 shows that -1 peptide and anti-CD28 immunobeads were used to culture umbilical cord blood-derived CD8-positive cells after WT-1 peptide specific CTL induction, with FITC-labeled anti-CD3 antibody and PE-labeled HLA-A*24:02WT-l ( Mu) - Quaternary and APC-labeled anti-CD8 antibody staining - and the results of one-dimensional flow cytometry analysis of the intensity of the light wavelength of pe and APC among cells passing through CD3+ gates. Figure 4A shows that the results are not applied. Cord blood-derived CD8-positive cells cultured with CTL induction treatment, stained with FITC-labeled anti-CD3 antibody, stained with APC-labeled anti-CD8 antibody, and measured by the CD3+ gate, the fluorescence wavelengths of pe and APC were measured. Results of two-dimensional flow cytometry analysis. Figure 4B shows cord blood-derived CD8-positive cells cultured without CTL induction treatment, FITC-labeled anti-CD3 antibody, and PE-labeled HLA-A*24:02WT-l (mu)-Quaternary Body and APC Labeling Anti-CD8 Body staining, and the results of two-dimensional flow cytometry analysis of the intensity of fluorescence of PE and APC at the wavelength of 201245224 were measured by cells of CD3+ gate. Figure 4C shows stimulation with hTERT soluble peptide and anti-CD28 immunobeads. Post-cultured peripheral blood-derived CD8-positive cells were stained with FITC-labeled anti-CD3 antibody, labeled with PE-labeled HLA-A*24: 02hTERT-quaternary and APC-labeled anti-CD8 antibodies, and assayed by CD3+ gate cells. Results of two-dimensional flow cytometry analysis of the intensity of fluorescence wavelengths of PE and APC. Figure 4D shows that FITC-labeled anti-CD3 antibody and APC-labeled anti-CD8 antibody were mixed and stained without mixing PE labeled hTERT peptide tetramer. The resulting sample was subjected to a two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of the cells at PE and APC by CD3+ gate. Figure 5A shows CD8-positive cord blood derived from culture without CTL induction treatment. A two-dimensional flow cytometer that measures the intensity of the fluorescence wavelength of pe and APC among cells stained with F ITC, anti-CD3 antibody, and APC-labeled anti-CD8 antibody, and passed through CD3+ gates. The results of the analyzer analysis. Figure 5B shows CD8-positive cells derived from cord blood cultured without CTL induction treatment, FITC-labeled anti-CD3 antibody, and PE-labeled HLA-A*24:02survivin-2B-quaternary body The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of PE and APC were measured by staining with APC-labeled anti-CD8 antibody and passing through CD3+ gates.

Figure 5C shows CD8-positive cells from the end of blood-stained cells stimulated with HLA-A*24:02 survivin-2B AYACNTSTL soluble peptide and anti-CD28 immunobeads, labeled with FITC-labeled anti-CD3 antibody, and pe 40 201245224 labeled HLA- A*24: The result of two-dimensional flow cytometry analysis in which the intensity of the fluorescence wavelength of PE and APC was measured by staining the 02hTERT-quaternary body with the APC-labeled anti-CD8 antibody and passing through the CD3+ gate. Figure 5D shows the sample obtained by mixing the FITC-labeled anti-CD3 antibody and the APC-labeled anti-CD8 antibody without mixing the PE-labeled survivin-2B peptide-quaternary body, and the sample was passed through a CD3+ gate to measure the fluorescence wavelength of the cells in PE and APC. The strength of the two-dimensional flow cytometer is analyzed as a result. Figure 6A shows umbilical cord blood-derived CD8-positive cells cultured with WT-1 mutant peptide, CMVpp65 peptide, hTERT soluble peptide, survivi η-2B soluble peptide and anti-CD 2 8 immunobeads, with FITC Labeling anti-3} antibody, stained with 标记£111111*4:24#02#1'-1 (1111〇-quaternary and octa-labeled anti-CD8 antibody, and measured by CD3+ gates in cells The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of ΡΕ and APC. Figure 6Β shows immunity against WT-1 mutant peptide, CMVpp65 peptide, hTERT soluble peptide, survi vin_2B soluble peptide and anti-CD28 Umbilical cord blood-derived CD8-positive cells cultured after bead stimulation were stained with FITC-labeled anti-CD3 antibody, pe-labeled HLA-A*24: 02CMVpp65-quaternary body and APC-labeled anti-CD8 antibody, and determined by cells in the gate Results of two-dimensional flow cytometry analysis of the intensity of fluorescence wavelengths of PE and APC. Figure 6C shows immunity against WT-1 mutant peptide, CMVpp65 peptide, hTERT soluble peptide, survivin_2B soluble peptide and anti-CD28 Cultured after bead stimulation CD8-positive cells with blood-derived antibodies, labeled with FITC-labeled anti-41 201245224 匸03 antibody, and with the mark of 111^4*24:0 2 butyl-tetra-tetramer and 4?0:5-anti-CD 8 antibody The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of PE and APC among the cells stained and passed through the c D 3+ gate. Figure 6D shows the peptide with the WT-1 mutation, CMVpp65 peptide , hTERT soluble peptide, survivin-2B soluble peptide and anti-CD28 immunobead stimulated cord blood-derived CD8-positive cells, FITC-labeled anti-CD3 antibody, and PE-labeled HLA-A*24:02survivin-2B-four The results of two-dimensional flow cytometry analysis of the intensity of the fluorescence wavelength of PE and APC in the cells stained with the APC-labeled anti-CD8 antibody and passed through the CD3+ gate. Figure 6E shows that CTL induction was not performed. Control experiments of cord blood-derived CD8-positive cells were stained with F1TC-labeled anti-CD3 antibody, with PE-labeled HLA-A*24:02 WT-l(mu)-quaternary and APC-labeled anti-CD8 antibodies, and passed through CD3+ gates. A two-dimensional flow measuring the intensity of the fluorescence wavelength of pe and APC among cells The result of the analysis by the cytokine counter. Figure 6F shows the intensity of the fluorescence wavelength of PE and APC measured by passing the FITC-labeled anti-CD3 antibody and the sample stained with the APC-labeled anti-CD8 antibody through the CD3+ gate. The results of flow cytometric analysis. [Key device symbol description] 9 CD positive cells 10 Soluble peptide 11 Solid support 42 201245224 12 Anti-CD28 antibody 13 CD28 molecule 14 T cell receptor / CD3 / CD8 complex 15 HLA class I molecule 16 HLA class I Molecular cells 9 recognize cytotoxic T cells of soluble peptide 2 restricted by HLA class I molecule 7 201245224 Sequence Listing <110> National University Corporation Tokyo University <110>Tai Lai Co., Ltd. <120> Composition for inducing cytotoxic T cells <130> 39179 <160> 5 <170> Patentln version 3. 3 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <;220>

<221> MISC.FEATURE <223> HLA-A*0201 WT1 (residues 235-243) <400〉 1

Arg Met Phe Pro Asn Ala Pro Tyr Leu 1 5 &lt;210〉 2 <211> 9

&lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220〉

&lt;221&gt; MISC_FEATURE &lt;223&gt; HLA-A*A2402 WT1 (mutant) (residues 235-243); 235-243 of human WT1 where the methionine (M) residue at 236 is replaced with tyrosine (Y). &lt; 400> 201245224

Cys Tyr Thr Trp Asn Gin Met Asn Leu 1 5 &lt;210&gt; 3 &lt;211〉 9

&lt;212> PRT &lt;213&gt; Homo sapiens &lt;220〉

&lt;221> MISC_FEATURE &lt;223&gt; HLA-A*2402 hTERT (residues 461-469) &lt;400&gt; 3

Val Tyr Gly Phe Val Arg Ala Cys Leu 1 5 &lt;210〉 4 &lt;211〉 9

&lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; MISC_FEATURE &lt;223&gt; HLA-A*2402 survivin-2B (residues 80-88) &lt;400&gt; 4

Ala Tyr Ala Cys Asn Thr Ser Thr Leu 1 5

&lt;210> 5 &lt;211> 9 &lt;212> PRT &lt;213> Human cytomegalovirus &lt;220&gt;&lt;221> MISC FEATURE 201245224 &lt;223&gt; HLA-A*2402 HCMV pp65 (residues 341-349) &lt;;400&gt; 5

Gin Tyr Asp Pro Val Ala Ala Leu Phe

Claims (1)

201245224 VII. Patent Application Range: 1. A composition for inducing cytotoxic tau cells, characterized by 'containing an anti-CD28 antibody, a solid support having the anti-CD28 antibody immobilized, and being compatible with MHC I A soluble peptide that binds to a molecule. 2. The composition for inducing cytotoxic tau cells as described in claim 1, wherein the solid support is a cell culture vessel. 3. The composition for inducing cytotoxic T cells according to claim 1 or 2, wherein the soluble peptide which binds to the mhc class I molecule is Hla compatible by the patient. The HLA complex is suggested as an antigen and is recognized by the cytotoxic T cells. A pharmaceutical composition for treating a tumor, which comprises the composition for inducing cytotoxic tau cells as described in claim 3, and which is soluble in the MHC class I molecule. The peptide comprises a partial amino acid sequence of a specific antigenic protein of the tumor, which is recognized by the cytotoxic T cell. 5. The pharmaceutical σ substance for treating a tumor as described in claim 4, wherein the tumor cell has a specific antigenic protein of 1. A pharmaceutical composition for treating a viral disease, characterized in that, as described in claim 3, the method for inducing cytotoxic tau cells is as described in the third paragraph of the patent application. The soluble fraction of the molecular-like molecule comprises a partial amino acid sequence of a specific antigenic protein of the virus, and the cytotoxic sputum cell recognizes the virus-infected cell. 7. The versatile antigenic protein of the virus for use in the treatment of viral diseases as described in claim 6 of the patent application, wherein the specific antigenic protein of the virus is the pp65 protein of the mega 201245224 virus. 8. A pharmaceutical composition comprising a cytotoxicity τ obtained by exposing a composition for inducing cytotoxic tau cells according to claim 1 or 2 to CD8 positive cells derived from hematopoietic stem cells. The cytotoxic T cell recognizes the cytotoxic T cell of the soluble peptide which binds to the MHC class J molecule. 9. The pharmaceutical composition according to claim 8, wherein the hematopoietic stem cell line is selected from the group consisting of embryonic stem cells, adult stem cells, and artificial pluripotent stems (ips). Any of the stem cell-derived hematopoietic stem cells, cord blood-derived hematopoietic stem cells, blood-derived hematopoietic stem cells, and bone marrow blood-derived hematopoietic stem cells. A method for producing a pharmaceutical composition comprising a cytotoxic steroid, a cell, comprising the steps of: (1) giving advantages of CD8-positive cells from hematopoietic cells selected from at least one of the group consisting of: The step of multiplication: a hematopoietic stem cell derived from any group of stem cells derived from embryonic stem cells, adult stem cells, and artificial pluripotent sul- p committed dry (lPS) cells, . The hematopoietic stem of the source of blood at the end of the stem is ^ Α Η .* . The stem cells of the stem cells of the cell and bone marrow, (2) the toxicity as described in item i or 2 of the patent scope The T cell composition is exposed to the (10) positive cells, and the field (3) is cultured to identify the step of binding the MHC class I molecule to the cytotoxic T cells. The present invention is a method for producing a pharmaceutical composition comprising cytotoxic T cells according to claim 10, wherein the soluble peptide which binds to the MHC class I molecule comprises human WT- 1 amino acid or cytomegalovirus amino acid sequence of ρρ65 protein.