JP2007246451A - Cancer vaccine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide capable of efficiently inducting tumor immunization, a composition containing the peptide, an antigen-presenting cell presenting an antigen originated from the peptide, a cytotoxic T cell stimulated with the antigen-presenting cell, a cancer vaccine utilizing the peptide and the cell, and a method for treating a tumor. <P>SOLUTION: A peptide having a specific sequence comprising about nine amino acids, a composition containing the peptide, an antigen-presenting cell, such as dendritic cells, presenting an antigen originated from the peptide, or a T-cell, such as a cytotoxic T-cell, activated with the antigen-presenting cell, is made, and the peptide, the antigen-presenting cell, or the cytotoxic T-cell is administered into a tumor patient as a vaccine. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cancer vaccine.

  In recent years, the mechanism of tumor antigen recognition by immune cells has been elucidated considerably in tumor immunology. According to this, first, dendritic cells (DCs), which are antigen-presenting cells, express an antigenic peptide consisting of 8 to 10 amino acids generated when degrading a protein expressed by a tumor in the cell, Presented on the cell surface together with a major histocompatibility complex or MHC (human leukocyte antigen or HLA in humans). Cytotoxic T cells (cytotoxic T lymphocytes or CTLs) recognize antigen peptides bound to HLA class I on the surface of dendritic cells, activate and proliferate, invade tumors, and produce proteins derived from antigen peptides It causes cytotoxicity against tumor cells (Arch. Surg. (1990) 126: 200-205).

  By utilizing this mechanism, cancer vaccines have been developed as a method for treating tumors. For example, dendritic cells that present tumor-specific protein-derived antigenic peptides on the cell surface are generated in vitro, proliferated, administered to tumor patients, or cytotoxic T cells educated by the dendritic cells. In other words, tumor immunity is induced in the body of a tumor patient. Alternatively, a tumor-specific protein is administered to a tumor patient and induces the whole process of the tumor immune mechanism in the patient's body (Science (1991) 254: 1643-1647; Exp. Med. (1996) 183: 1185 -1192; J. Immunol. (1999) 163: 4994-5004; Proc. Natl. Acad. Sci. USA (1995) 92: 432-436; Science (1995) 269: 1281-1284; J. Exp. Med. (1997) 186: 785-793).

  Results from clinical trials have been reported for melanoma-specific tumor antigens. For example, tumor reduction has been observed in 42% of patients by subcutaneous administration of melanoma antigen gp100 peptide to melanoma patients and intravenous administration of interleukin-2 (Nature Medicine (1998) 4: 321).

  However, only a few examples of tumor-specific proteins that can efficiently induce tumor immunity are known in some tumors.

  Therefore, the present invention provides a peptide capable of efficiently inducing tumor immunity, a composition containing the peptide, an antigen-presenting cell presenting the peptide, a T cell stimulated by the antigen-presenting cell, and these peptides and cells The purpose of the present invention was to provide a cancer vaccine utilizing the above and a method for treating tumor patients using the same.

  The present inventors have already found that the transcription factor SOX6 is expressed in glioma (Ueda et al., Oncogene 23, 1420-1427, 2004), but is also expressed in other tumors. (See Example 2). Based on this finding, it was investigated whether various partial peptides of SOX6 were recognized by cytotoxic T cells in an HLA class I-restricted manner. When they were added to dendritic cells, they were efficiently cytotoxic T cells. Several partial peptides recognized by can be identified, and the present invention has been completed.

  The peptide according to the present invention has the sequence of SEQ ID NO: 10. Antigen-presenting cells presenting this peptide and T cells that are induced by this antigen-presenting cell and recognize cells that express SOX6 also belong to the technical scope of the present invention. This T cell is preferably a cytotoxic T cell, and cells expressing SOX6 are glioma, liver cancer cell, lung cancer cell, pancreatic cancer cell, esophageal cancer cell, melanoma cell, prostate cancer cell, breast cancer cell, renal cancer cell. Or a leukemia cell.

  Furthermore, the cancer vaccine according to the present invention includes one or more peptides selected from a peptide having the sequence of SEQ ID NO: 10 and / or a peptide having the sequences of SEQ ID NOs: 1 to 3, the antigen-presenting cell, and the T cell. Among them, at least one is contained. This cancer vaccine is a cancer vaccine against cancer cells expressing SOX6, and is particularly preferably a cancer vaccine against glioma, liver cancer, lung cancer, pancreatic cancer, esophageal cancer, melanoma, prostate cancer, breast cancer, kidney cancer, or leukemia.

  The tumor treatment method according to the present invention is characterized by using this cancer vaccine against humans and non-human vertebrates.

  According to the present invention, a peptide capable of efficiently inducing tumor immunity, a composition containing the peptide, an antigen-presenting cell presenting the peptide, a T cell stimulated by the antigen-presenting cell, and these peptides and cells are used. It has become possible to provide a cancer vaccine and a method for treating a tumor patient using the same.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Screening for peptides with high binding to HLA class I ==
A 9-10 amino acid residue peptide with high binding to HLA class I from the amino acid sequence encoded by the SOX6 gene using the HLA Peptide Binding predictions Program of BioInformatics & Molecular Analysis Section (BIMAS), a database on the website The sequence was identified. Japanese HLA class I types are mostly HLA-A2 and HLA-A24, which cover about 80% of all Japanese people. In addition, HLA-A2 includes A0201 and A0206, and HLA-A24 includes A2402 as HLA-A genotypes common in Japanese. In this specification, when the cell genotype is described as A0201, A2402, Means having at least one allele.

Here, screening was performed for binding to A24. Table 1 shows examples of high-scoring peptide sequences obtained by this screening.

  When these peptides are actually synthesized and administered as antigens on the surface of dendritic cells, which are antigen-presenting cells, they are presented on the cell surface by binding to HLA class I molecules and recognized by cytotoxic T cells. In order to identify peptides capable of inducing specific cytotoxic T cells, the reactivity of cytotoxic T cells co-cultured with dendritic cells to human glioma cells was examined. As a result, dendritic cells presenting the peptides of SEQ ID NOs: 1 to 3 efficiently stimulate cytotoxic T cells, and cytotoxic T cells established by stimulation of each peptide express SOX6. It was revealed that glioma cells were recognized efficiently.

  In the following examples, by showing the results of these experiments, when the peptides of SEQ ID NOs: 1 to 3 are added to antigen-presenting cells, the peptides are presented on HLA and stimulate co-cultured T cells. Induces antigen-specific T cells that react with glioma cells that express SOX6 or are cytotoxic to cells that express SOX6. From this, the peptide of SEQ ID NOs: 1 to 3, the antigen-presenting cell presenting the peptide on the cell surface, and the cytotoxic T cell established by stimulation of the antigen-presenting cell are useful as a cancer vaccine against glioma. I understand that.

Furthermore, when a mutation was introduced into the sequence of SOX6-504 (SEQ ID NO: 1: RFENLGGPQL), one of the peptides thus identified, the binding affinity for HLA-A2402 and the like were examined. As shown in the Examples, cytotoxic T cells stimulated by peptide SOX6-504M (Table 2) having the sequence of SEQ ID NO: 10 (RYENLGGPQL) were stimulated by SOX6-504 and induced cytotoxicity. It was revealed that proliferative ability and cytotoxic activity are higher than those of sex T cells.

  Therefore, a peptide having the sequence of SEQ ID NO: 10, an antigen-presenting cell presenting the peptide on the cell surface, and a cytotoxic T cell established by stimulation of the antigen-presenting cell are useful as a cancer vaccine against glioma.

== Method of administration of cancer vaccine ==
Currently, methods for administering tumor-specific cancer antigens, cancer antigen-presenting antigen-presenting cells, or cancer antigen-reactive cytotoxic T cells to tumor patients are being developed as cancer vaccines. In the present invention, since a partial peptide of SOX6 is used, the tumor to be treated (including prevention) may be any tumor that expresses SOX6, and particularly expresses SOX6 at a high level. Other gliomas, liver cancer, lung cancer, pancreatic cancer, esophageal cancer, melanoma, prostate cancer, breast cancer, kidney cancer, or leukemia are also treated. The main treatment target is a human patient having such a tumor, but it may be a vertebrate other than a human having a tumor.

  A cancer vaccine to be administered to a patient having a tumor to be treated may contain a peptide having SEQ ID NO: 1 to 3 or 10 that can be a tumor-specific cancer antigen. In this case, the patient's HLA class I type is examined in advance and a suitable peptide is used. Here, when the patient's HLA class I type is A24, the peptide of SEQ ID NO: 1 to 3 or 10 is administered. The peptide to be administered may be one type or a plurality of types. Regarding the administration site, intradermal administration, subcutaneous administration, intravenous administration, intraperitoneal administration, and the like can be considered, and there is no particular limitation. Moreover, when administering, you may administer a peptide with the adjuvant etc. which improve immunity induction ability. Moreover, the peptide to be administered may be modified so as not to be degraded in vivo.

  Further, the cancer vaccine may contain antigen-presenting cells presenting peptides having SEQ ID NOs: 1 to 3 or 10. Here, the peptide displayed on the cell surface may be the peptide itself having SEQ ID NO: 1 to 3 or 10, or may be modified with sugar, phosphoric acid or the like. In addition to dendritic cells, macrophages, B cells, and the like can be considered as antigen-presenting cells. Dendritic cells are preferred because of their high antigen-presenting ability. Hereinafter, an example of a method for isolating dendritic cells will be described.

  First, mononuclear cells are isolated from the peripheral blood of vertebrate individuals. The mononuclear cells are preferably separated from the individual to be treated, but may be isolated from other individuals. The mononuclear cells are preferably CD14 positive. When the isolated mononuclear cells are cultured for about 7 days with GM-CSF and IL-4, they can be induced to differentiate into immature dendritic cells. The dendritic cells thus induced to differentiate highly express MHC molecules that are antigen-presenting molecules. The immature dendritic cell is examined for HLA class I type, and if it is A24, the peptide of SEQ ID NO: 1 to 3 or 10 is added. What is added is not limited to an artificially synthesized peptide, but may be an extract (extract or lysate) of a cell in which the peptide is expressed or a purified product. The antigen-presenting dendritic cells thus obtained are administered to an individual having a tumor. With regard to the administration site, intradermal administration, subcutaneous administration, intravenous administration, intralymphatic administration, etc. are conceivable, and although there is no particular limitation, physiological dendritic cell antigen presentation is not limited to dendritic cell administration site. In view of the fact that it is performed in the regional lymph nodes, direct administration into the lymph nodes is preferable.

  The cancer vaccine may contain T cells established by stimulation of antigen-presenting cells presenting peptides derived from SEQ ID NOs: 1-3 or 10. T cells are co-cultured with antigen-presenting cells presenting peptides derived from SEQ ID NOs: 1-3 or 10 and stimulated with antigen-presenting cells. The T cells thus established may be administered to an individual having a tumor. The T cell here is preferably a cytotoxic T cell, but may be a helper T cell or the like. Regarding the administration site, intradermal administration, subcutaneous administration, intravenous administration, intratumoral administration, and the like are conceivable. Although there is no particular limitation, in the case of cytotoxic T cells, cells expressing the antigen can be directly attacked. Therefore, intratumoral administration is preferred.

<Example 1> Activation of cytotoxic T cells by dendritic cells presenting antigen == First day of experiment ==
Mononuclear cells were isolated from peripheral blood of healthy individuals with HLA class 1 type A24 (genotype is A2402) as follows. First, 50 ml of peripheral blood was collected with a syringe washed with 5 ml of heparin. An equal amount of Lymphoprep (Fresenius kabi Norge AS, Axis-Shield PoC AS, Oslo, Norway) was mixed by inversion, centrifuged (20 ° C., 2000 rpm, 35 minutes, without brake), and the intermediate layer was sucked and collected. PBS was added to re-turbidize, and the operation of centrifuging (20 ° C., 2000 rpm, 10 minutes) was repeated 3 times to wash the resulting mononuclear cells.

The mononuclear cells were seeded in a culture dish for primary cell culture (FALCON MULTIWELL PRIMARIA 24 well) at a density of 5 × 10 ⁶ cells / well and cultured at 37 ° C. in the presence of 5% CO ₂ for 4 hours. The culture solution used was a mixture of AIM-V (GIBCO) and RPMI-1640 in 1: 1 (basic culture solution). Cells adhering to the bottom of the culture dish were collected, seeded in a new 24-well culture dish at a density of ⁴ × 10 ⁵ cells / well, and GM-CSF (10 ng / ml), IL-4 (1 ng / ml) added Using the culture solution, the cells were cultured for 7 days at 37 ° C. in the presence of 5% CO ₂ to differentiate the dendritic cells.

== Experiment day 7 ==
Dendritic cells differentiated on the seventh day of culture were used as antigen presenting cells (APCs). After the obtained dendritic cells were irradiated (60 Gy), 10 μM of the synthesized and purified peptide (the peptide derived from SOX6 of SEQ ID NOs: 1 to 3 and 5) was added, and the mixture was incubated at 37 ° C. in the presence of 5% CO ₂ for 2 hours. The cells were cultured and bound to cells, washed twice with PBS, and used as stimulator cells. As a control, an experiment using a cytomegalovirus (CMV) -derived peptide (see SEQ ID NO: 4: QYDPVAALFF, Provenzano et al., Transfusion 43, pp1567-1574) instead of a SOX6-derived peptide was performed.

On the other hand, in order to obtain dendritic cells on this day (the 7th day of culture) and the 15th day of experiment, mononuclear cells were cultured by the same operation as the first day.
In the process, after 4 hours of culture Mononuclear cells, it does not adhere to the culture dish, the cells floating in the culture supernatant was collected, IMag anti-human CD8 to 1x10 ⁷ cells particules-DM (BD Biosciences) 100 μl was reacted at 4 ° C. for 30 minutes. CD8 positive cells were adsorbed and collected using a magnet, and used as responder cells.

2 × 10 ⁵ responding cells thus obtained were added to the wells of the stimulating cells and co-cultured. In addition, IL-1α (10 unit / ml), IL-2 (20 unit / ml), IL-4 (1 ng / ml), IL-6 (125 unit / ml), IL-12 ( A basic culture solution supplemented with 1 ng / ml) was used.

== Experiment 15 day ==
Stimulatory cells were prepared in the same manner as described above using dendritic cells cultured and differentiated from day 7 of culture.
Moreover, the response cells co-cultured with the stimulator cells were collected from the 7th day of culture, and co-cultured with the newly prepared stimulator cells in the same manner. However, the culture solution used was a culture solution not containing IL-12 in the culture solution used on the seventh day of culture.
In addition, the culture of mononuclear cells to obtain dendritic cells on the 22nd day of the experiment was newly started in the same manner.

== Experiment 22 day ==
As in the 15th day of the experiment, the responder cells that had been co-cultured with the stimulator cells were collected and co-cultured with the newly prepared stimulator cells.
In addition, although the culture | cultivation of the mononuclear cell for obtaining a dendritic cell was started, it seeded | inoculated and adjusted to ⁴ * 10 < ⁴ > piece / well using a 96-well culture dish instead of a 24-well culture dish.

== Experiment 29th day ==
Dendritic cells differentiated using mononuclear cells cultured in 96 wells were subjected to 0 μM, 0.1 μM, 1 μM, 10 μM peptides derived from SOX6, or 0 μM, 0.1 μM, 1 μM, 10 μM concentrations. A peptide derived from CMV was added, and the cells were cultured for 2 hours at 37 ° C. in the presence of 5% CO ₂ to bind to the cells, washed twice with PBS, and used as target cells.
Responding cells that continued to co-culture were collected, and cells adjusted to 2 × 10 ⁴ as effector cells were added to the wells of the newly prepared target cells and co-cultured.

== Experiment 31st day ==
The culture supernatant was collected from the co-culture and subjected to IFN-γ ELISA as follows.
On the previous day, the primary antibody (anti-human IFN-γ monoclonal Ab, Endogen) was diluted to 0.5 μg / ml with PBS, and 100 μl per well was added to a 98-well plate (Nunc Maxisorp). The plates were coated overnight. On the day of the experiment, 300 μl per well of 5% FCS-containing PBS was treated for 1 hour at room temperature for blocking. The wells were washed 3 times with PBS containing 0.2% Tween-20, 50 μl of culture supernatant recovered from co-culture was added, and biotinylated secondary antibody (0.5 mg / ml with PBS containing Endogen, 5% FCS) was added. 50 μl) was added and allowed to stand at room temperature for 2 hours. After the wells were washed 3 times with PBS containing 0.2% Tween-20, 100 μl of streptavidin-conjugated HRP (ExtraAvidine peroxidase conjugate, SIGMA E-2886, diluted 4000 times with PBS containing 5% FCS) was added, Left for 30 minutes. The wells were washed 3 times with PBS containing 0.2% Tween-20 and then developed with TMB as a substrate (TMB was prepared by dissolving 1 tablet of TMB tablet in 1 ml of DMSO and adding 9 ml of phosphate citrate buffer. ). 25 μl of 1N H ₂ SO ₄ was added to stop the reaction. The absorbance at 450 nm was measured with a plate reader, the concentration of IFN-γ was calculated from a standard curve prepared in advance with a known concentration of IFN-γ, and the results are shown in FIG.

== Result ==
Of SOX6-derived peptides predicted to be able to bind to HLA-A24 (see Table 1), cells stimulated by co-culture with dendritic cells supplemented with SOX6-360, SOX6-504, and SOX6-628 When T-cells (effector cells) were co-cultured with dendritic cells presenting the respective antigen peptides as targets, the amount of IFN-γ released increased compared to various controls (FIG. 1). This is because, when peptides derived from SOX6 peptides (SOX6-360, SOX6-504, and SOX6-628) are added to antigen-presenting cells, antigen-presenting cells that present those peptides are obtained, and the obtained antigen presentation It shows that T cells specifically recognizing each peptide were induced by stimulating T cells with cells.

<Example 2> Reactivity of cytotoxic T cells established by stimulation with SOX6-derived peptides to SOX6-expressing cells [1] Expression analysis of SOX6 Specifically, by clarifying cells expressing SOX6, It was examined which cells are the target of the method of the present invention.

== Analysis by RT-PCR ==
Normal human tissues (adult brain and testis, and human fetal brain), glioma cell lines (GI-1, U87, and T98G), glioma tissues isolated from patients (glioblastoma tissue, and astrocytoma tissue), Quantification of SOX6 mRNA expression in human tumor cell lines (888mel, 928mel, 586mel, LK2, PC9, LU99, RERF-LC-MA, TE8, PK56, PK1, PC3, MDA231, RCC6, RCC8, KU7, and Molt4) Analyzed by dynamic PCR.

  Cells as material for isolating RNA were obtained as follows. First, glioma cell lines SF126 and Marcus were purchased from Health Science Research Resources Bank (Osaka, Japan). U-87-MG and T98G (glioma), GI-1 (gliosarcoma), 888mel, 928mel and 586mel (melanoma) Molt4 (leukemia and lymphoma), PC9, LU99, LK2, RERF-LC-MA (lung cancer), RCC6 and RCC8 (renal cell cancer), TE8 (esophageal cancer), PK1 and PK59 (pancreas cancer), and MDA231 (breast cancer) were purchased from American Type Culture Collection (Manassas, VA). In addition, glioma tissue was obtained from tumor specimens of surgical patients who had consented to the explanation text approved by Keio University Ethics Committee (No. 12-21-2). Total RNA was isolated from these cell lines or tissues using RNeasy (Qiagen). In addition, total human normal tissue RNA was purchased from CLONTECH Laboratories, Inc. These total RNAs were subjected to reverse transcription using AMV Reverse transcriptase XL (Takara Bio Inc., Ohtsu, Japan) and oligo dT primers to synthesize cDNA.

These cDNAs were subjected to quantitative PCR as follows to examine the expression of SOX6 mRNA. Primers for SOX6
forward primer, 5'-GATCGCCATCAACTCCACAGC-3 '(SEQ ID NO: 6)
reverse primer, 5'-GCTGCAGAGCCCATTCATGC-3 '(SEQ ID NO: 7)
The primer for β-actin as an internal control is
forward primer, 5'-GGCACCCACACAATGAAG-3 '(SEQ ID NO: 8)
reverse primer, 5'-GCCGATCCACACGGAGACTACT-3 '(SEQ ID NO: 9)
Was used. Quantitative PCR was performed at 95 ° C. for 10 minutes, followed by 50 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 1 minute, and finally extension reaction at 72 ° C. for 1 minute. The results are shown in FIG. 2A. In addition, the expression level of SOX6 in each tissue was standardized by the expression level of β-actin in each tissue.

== Analysis by Western blotting ==
Next, the expression of SOX6 at the protein level was examined by Western blotting. First, the cells obtained in the same manner as above were collected, put into a Dounce homogenizer together with a disruption buffer (final concentration 20 mM HEPES, final concentration 0.25 M sucrose added to 950 ml of pure water and adjusted to PH 7.5), and placed on ice. The cells were disrupted with The disrupted solution was centrifuged at 1000 g for 7 minutes, the supernatant was discarded, and the precipitate containing nuclei and unbroken cells was collected. An SDS gel loading buffer was added to the precipitate and dissolved to prepare a sample for electrophoresis. These samples were run in an amount of 20 μg in each lane using SDS-PAGE. SOX6 protein was recognized by anti-SOX6 antibody (CHEMICON) prepared to 10 μg / ml and reacted with AP-conjugated goat anti-rabbit IgG (Fc) antibody (2000-fold diluted, Cappel, Aurora, Ohio) Subsequently, color was developed with a mixture of Nitro blue tetrazolium (Boehringer Mannheim, GmbH, Germany) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, MO). The results are shown in FIG. 2B.

== Result ==
SOX6 is expressed not only in human fetal brain, adult testis, glioma cell line, glioma tissue, but also melanoma, small cell lung cancer, non-small cell lung cancer, squamous lung cancer, esophageal cancer, pancreatic cancer, prostate Expression was also detected in cell lines derived from cancer, breast cancer, kidney cancer, chronic myeloid leukemia, and T cell leukemia. (FIG. 2A).

  On the other hand, the expression of SOX6 protein in normal tissues was not detected except in testis (FIG. 2B). Furthermore, SOX6 is a protein not only in glioma cell lines (Marcus, U87, KNS-42, SF126) but also in lung cancer cell lines (PC9), hepatocellular carcinoma lines (HepG2) and pancreatic cancer lines (Panc-1). Expression was also detected at the level.

  As described above, SOX6 was detected only in testis in normal human adult tissues, but was widely detected in tumor cells. This indicates that a cancer vaccine using a peptide derived from SOX6 is effective not only for glioma but also for various tumors. Furthermore, since almost no expression is detected in normal tissues, this cancer vaccine is considered to have few side effects.

[2] Evaluation of reactivity to SOX6-expressing cells by the amount of IFN-γ released from effector cells == Experiment 29th day ==
Up to this date, effector cells were prepared in the same manner as in Example 1. In addition, the HLA class 1 type of a healthy person who obtains mononuclear cells is A24 (genotype is A2402), the peptide of SEQ ID NOs: 1 to 3 is used as the added peptide, and the CMV-derived peptide (SEQ ID NO: SEQ ID NO. 4: QYDPVAALFF).

On the other hand, human glioma was added at 37 ° C. in the presence of 5% CO ₂ using a culture solution to which mitomycin (200 pg / ml) was added (AIM-V (GIBCO) and RPMI-1640 mixed 1: 1). Cells KNS-81 (cells expressing both HLA-A24 and SOX6 but not HLA-A2) were cultured for 60 minutes, washed 5 times with PBS, and then incubated in a 96-well culture dish (COSTER 3595 -96 well) at 5 × 10 ³ cells / well and seeded as target cells.

For this target cell, 0 effector cells (in the graph, effector cell: target cell (E / T ratio) = 0), 5 × 10 ⁴ (E / T ratio = 10), 1 × 10 ⁵ (E / T ratio) = 20) Each of 2 × 10 ⁵ (E / T ratio = 40) was added and co-cultured.

  As controls, instead of human glioma cell KNS-81, CIR-24 cells (cells in which HLA-A24 is expressed but SOX6 is not expressed) and glioma cells U87 (in which HLA-A2 and SOX6 are both expressed) Experiments were carried out using cells that were expressed but not HLA-A24.

== Experiment 31st day ==
The culture supernatant was collected from the co-culture and subjected to IFN-γ ELISA. The results are shown in FIG.

== Result ==
Cytotoxic T cells (effector cells) induced with partial peptides derived from SOX6 (SOX6-504 and SOX6-628) respond to target glioma cells (KNS-81) and serve as controls. In comparison, the amount of IFN-γ released increased in a cell number ratio dependent manner (FIG. 3). This is because T cells established by stimulation with antigen-presenting cells presenting SOX6-504 or SOX6-628 are glioma cells (KNS-81) expressing SOX6 and HLA-24. It shows that it can be specifically recognized. Since the established T cells do not react with U87 (a cell in which both HLA-A201 and SOX6 are expressed but not HLA-A24), these SOX6-specific T cells are HLA-A24-restricted antigens. Was shown to recognize.

[3] Evaluation of reactivity to SOX6-expressing cells based on cytotoxicity of effector cells == Experiment 28th day ==
Up to this date, effector cells were prepared in the same manner as in Example 1. The type of HLA class 1 of the person who obtains mononuclear cells was A24 (A2402), and the peptides of SEQ ID NOS: 1 and 2 were used as added peptides. In this example, as peripheral blood for obtaining mononuclear cells, not only healthy human peripheral blood (result is FIG. 4) but also glioma patient peripheral blood (result is FIG. 5).

  As target cells, CIR-A2402 in which HLA-A2402 cDNA was introduced into HLA-A, B-deficient human B cell line CIR, CIR-A2402-SOX6 in which SOX6 gene was introduced into CIR-A24, and HLA-A0201 into CIR. CIR-A0201-SOX6 introduced with cDNA and SOX6 gene, glioma cells expressing both HLA-A24 and SOX6 (Marcus, SF126, KNS-42), other cancers expressing both HLA-A24 and SOX6 Cells (hepatocellular carcinoma-derived HepG2, lung cancer-derived PC9) were prepared. The expression of SOX6 in CIR-A2402-SOX6 cells and CIR-A0201-SOX6 cells was confirmed by the Western blotting (FIG. 2B).

50 μl of 500 μl of Fetal Bovine Serum (FBS) and ⁵¹ Cr (1.85 MBq / 50 μl) was added to ^{6 of} 5 × 10 ⁶ cells, and the mixture was shaken and cultured at 37 ° C. under 5% CO 2 for 60 minutes. Performed subsequently washed 3 ^times, was added in 100 [mu] l to each well of a 96-well plate was adjusted to 5x10 3 / 100μl (COSTER 3595-96 well ).

To the target cells labeled with this ⁵¹ Cr, per well, 3x10 ⁵ cells of effector cells (E / T ratio = 60) , 1.5x10 5 cells (E / T ratio = 30) , 7.5X10 4 cells (E / T ratio = 15) was added and cultured at 37 ° C. in the presence of 5% CO ₂ for 4 hours.

The cpm (cpm experimental release) of each well was measured, and the specific solubility (Percentage of specific lysis) was calculated from the following equation.
Specific solubility = (cpm-csr) / (cmr-csr) x100
(Note that csr (cpm spontaneous release) is the cpm of the well added with the medium not containing the effector cell, and cmr (cpm maximal release) is the cpm of the well added with 0.1% Triton X not containing the effector cell. is there.)

== Result ==
CIR-A24-SOX6, glioma cell-derived Marcus, glioma cell-derived SF126, glioma cell-derived KNS-42, liver cancer-derived HepG2, and lung cancer-derived PC9 are cells expressing HLA-A24 and SOX6. As shown in FIG. 4 (when CTL is induced from peripheral blood lymphocytes derived from healthy individuals) and FIG. 5 (when CTL is induced from peripheral blood lymphocytes derived from glioma patients), SOX6-504 or SOX6-628 CTL (SOX6-504) and CTL (SOX6-628), which are cytotoxic T cells (effector cells) stimulated by coculture with dendritic cells, express these HLA-A24 and SOX6 Cells were lysed in an E / T ratio dependent manner.

  On the other hand, it has low solubility in cell CIR-A0201-SOX6 expressing SOX6 but not expressing HLA-A24 and cell CIR-A2402 expressing HLA-A24 but not expressing SOX6 From these results, it was shown that CTL (SOX6-504) and CTL (SOX6-628) can damage HLA-restricted cells that express SOX6 widely and specifically.

[4] Specificity of effector cells to target cells By examining the cytotoxic activity of effector cells established by stimulation with peptides derived from SOX6 on cells having peptides unrelated to SOX6 on the surface, the specificity of effector cells to target cells I examined the sex.

Basically, the experiment was performed in the same manner as in Example 2 [3].
(1) Glioma cell line Marcus expressing both HLA-A24 and SOX6
(2) CIR-A2402 (SOX6-504) obtained by binding peptide SOX6-504 having SEQ ID NO: 1 to CIR-A2402 expressing HLA-A24 but not SOX6 or peptide having SEQ ID NO: 2 CIR-A2402 combined with SOX6-628 (SOX6-628)
(3) CIR-A2402 (CMV) in which an unrelated peptide derived from CMV (SEQ ID NO: 4) is bound to CIR-A2402 as a control for measuring nonspecific cytotoxicity
(4) Untreated CIR-A2402
Was used. In the procedure described in Example 2 [3], labeled the cells with ^{51 Cr,} were added in 100 [mu] l to each well of a 96-well plate was adjusted to 5x10 3/100 [mu] l.

As effector cells, CTL (SOX6-504) established by stimulation with peptide SOX6-504 and CTL (SOX6-628) established by stimulation with peptide SOX6-628 were prepared. 1 × 10 ⁵ effector cells per well (ET ratio = 20: 1 was added to target cells on a 96-well plate. After culturing at 37 ° C. in the presence of 5% CO ₂ for 4 hours, cpm ( cpm experimental release) was measured, and the specific solubility was calculated from the above equation, and the results are shown in FIG.

  CTL (SOX6-504) and CTL (SOX6-628) are soluble in Marcus expressing both HLA-A24 and SOX6, and CIR-A2402 bound with SOX6-derived peptide SOX6-504 or SOX6-628. Was higher than the dissolution rate in CIR-A2402 to which a peptide which is an irrelevant peptide was bound or in untreated CIR-A2402. From this, it was shown that CTL (SOX6-504) and CTL (SOX6-628) specifically cause cell damage to cells presenting SOX6-504 and SOX6-628, respectively.

  Thus, in this Example, it was shown that the specificity of the effector cells induced with the SOX6-derived peptide to the target cells is high, and therefore, when the SOX6-derived peptide is used as a cancer vaccine, the side effects are low. it is conceivable that.

Example 3 Reactivity of Cytotoxic T Cells Established by Stimulation with Mutant-Induced SOX6-Derived Peptide to SOX6-expressing Cells [1] Cytotoxicity by Dendritic Cells Presenting Mutant-Induced Peptide SOX6-504M Evaluation of cell activation and proliferative ability Basically, cytotoxic T cells (CTL: effector cells) were induced by the same procedure as in Example 1. Similarly, peripheral blood from which a mononuclear cell is obtained is HLA class 1 type A2402 healthy human blood. As added peptides, SOX6-504 (SEQ ID NO: 1) and SOX6-504M (SEQ ID NO: 10) are used. It was.

== Measurement of CTL cell number change ==
In the process of peptide-specific CTL establishment, the number of cells per well of CD8 positive T cells adjusted to 2 × 10 ⁵ cells / well on day 7 was measured over time (14th day, 21st day, 28th day). did. Experiments were performed in three wells for each of CTL established by stimulation with SOX6-504 (SOX6-504) and CTL established by stimulation with SOX6-504M (SOX6-504M). When the cells were collected, CD8 positive T cells were isolated with IMag anti-human CD8 particules-DM, and the number of cells was measured. The results are shown in the graph of FIG.

[2] Evaluation of cytotoxic activity of cytotoxic T cells established by stimulation with SOX6-504M Basically, as described in Example 1, peptide-specific CTLs were induced, and Example 2 [3 The cytotoxic activity was measured by the same procedure as described above. As peripheral blood for obtaining mononuclear cells, peripheral blood of a healthy person having HLA class 1 type A2402 was used.

As target cells, CIR-A2402 in which HLA-A2402 cDNA was introduced into HLA-A, B-deficient human B cell line CIR, CIR-A2402 (CIR-A2402-SOX6) in which SOX6 gene was introduced into CIR-A2402 strain, CIR-A0201 (CIR-A0201-SOX6) introduced with SOX6 gene, glioma cells expressing both HLA-A24 and SOX6 (Marcus, SF126, KNS-42), PC9 cell line derived from lung cancer, HepG2 derived from liver cancer The strains were used, and the E / T ratio was tested in three ways: 20/1, 60/1, and 0/1. After co-culture at 37 ° C. in the presence of 5% CO ₂ for 4 hours, the cpm of each well was measured, and the specific solubility was calculated from the above formula. The results are shown in FIG.

Furthermore, peptide-specific CTLs derived from healthy human peripheral blood and peripheral blood of glioma patients were re-established, and CIR-A2402, CIR-A2402-SOX6, glioma cells (Marcus, SF126) were used 6 days before the above experiment. On the 22nd day, ⁵¹ Cr release cytotoxicity assay was performed. The results are shown in FIG. 9 (CTL derived from peripheral blood of a healthy person) and FIG. 11 (CTL derived from peripheral blood of a glioma patient).

== Result ==
SOX6-504-CTL and SOX6-504M-CTL derived from healthy human peripheral blood are cells in which both HLA-A24 and SOX6 are expressed, CIR-A2402-SOX6, Marcus, SF126, KNS-42, PC9, HepG2 was dissolved in an E / T ratio dependency. Further, the specific solubility thereof is the cell CIR-A0201-SOX6 expressing SOX6 but not expressing HLA-A2402, or the cell CIR-A2402 expressing HLA-A2402 but not expressing SOX6. SOX6-504-CTL and SOX6-504M-CTL were shown to be capable of causing specific cytotoxicity against HLA-restricted cells that express SOX6. FIG. 8).

Even when the ⁵¹ Cr release cytotoxicity assay was performed on the 22nd day, the same result as the measurement on the 28th day was obtained (FIG. 9). Using this data, the difference in specific solubility of CTL (SOX6-504M) and CTL (SOX6-504) with respect to the same target cells can be easily compared (FIG. 10). Thus, it is clear that the cytotoxic activity of CTL (SOX6-504M) is higher than that of CTL (SOX6-504).

  Similar results were confirmed in CTLs derived from peripheral blood lymphocytes derived from HLA-A2402-positive glioma patients (FIG. 11).

[3] Effect of CTL (SOX6-504M) induced by stimulation with SOX6-504M on SOX6-504 Basically, an experiment was performed in the same manner as in Example 2 [3].
(1) Glioma cell line Marcus expressing both HLA-A24 and SOX6
(2) CIR-A2402 (SOX6-504) in which peptide SOX6-504 (SEQ ID NO: 1) is bound to CIR-A2402 expressing HLA-A24 but not SOX6
(3) CIR-A2402 (CMV) in which an unrelated peptide derived from CMV (SEQ ID NO: 4) is bound to CIR-A2402 as a control for measuring nonspecific cytotoxicity
(4) Untreated CIR-A2402
Was used. In the procedure described in Example 2 [3], labeled the cells with ^{51 Cr,} were added in 100 [mu] l to each well of a 96-well plate was adjusted to 5x10 3/100 [mu] l.

As effector cells, CTL (SOX6-504M) established by stimulation with peptide SOX6-504M was prepared. 1 × 10 ⁵ effector cells per well (ET ratio = 20: 1) were added to target cells on a 96-well plate. These were cultured at 37 ° C. in the presence of 5% CO ₂ for 4 hours, cpm of each well was measured, and specific solubility was calculated from the above formula. The results are shown in FIG.

== Result ==
CTL (SOX6-504M) shows high specific solubility for Marcus and CIR-A2402 (SOX6-504) compared to specific solubilities for negative controls CIR-A2402 (CMV) and CIR-A2402. It was. From this, the CTL (SOX6-504M) induced by using the mutant peptide specifically recognizes cells presenting SOX6-504, which is a wild-type peptide originally presented by the tumor on HLA, and cytotoxicity. It was shown that it can cause

<Conclusion>
As described above, an antigen-presenting cell presenting the antigenic peptide can be produced using the peptide having SEQ ID NO: 10, and further, not only each peptide but also SOX6 was expressed by stimulation with the antigen-presenting cell. It has been shown that specific T cells that recognize the cells are induced.

In an example of the present invention, cytotoxic T cells (effector cells) induced by co-culture with dendritic cells to which a peptide derived from SOX6, which was expected to bind to HLA-A24, was added. -A graph showing the results of measuring IFN-γ released when co-cultured with dendritic cells presenting A24-binding SOX6-derived peptides as targets. In the Example in this invention, it is the figure which showed the result of the expression analysis of SOX6 by quantitative RT-PCT. In the Example in this invention, it is the figure which showed the result of the expression analysis of SOX6 by Western blotting. In the examples of the present invention, cytotoxic T cells (effector cells) induced by co-culture with dendritic cells to which a peptide derived from SOX6, which was expected to bind to HLA-A24, was added. It is a graph showing the result of measuring IFN-γ released when co-cultured with glioma cells (KNS-81) expressing A24 and SOX6 as targets. In the Examples of the present invention, cytotoxic T induced by co-culture with dendritic cells isolated from healthy human peripheral blood of HLA-A2402 and supplemented with SOX6-derived peptides SOX6-504 and SOX6-628. The specific solubility of target cells calculated when CIR-A2402, CIR-A2402-SOX6, Marcus, SF126, KNS-42, HepG2, and PC9 are co-cultured as target cells for cells (CTL: effector cells) is shown. It is a graph. In an example of the present invention, cytotoxic T induced by co-culture with dendritic cells isolated from peripheral blood of HLA-A2402 glioma patients and supplemented with SOX6-derived peptides SOX6-504 and SOX6-628. It is the graph which showed the specific solubility of the target cell calculated when coculturing CIR-A2402, CIR-A2402-SOX6, Marcus, and SF126 as a target cell with respect to a cell (CTL: effector cell). In Examples of the present invention, HLA-A2402 was isolated from healthy human peripheral blood and induced by co-culture with dendritic cells to which SOX6-derived peptide SOX6-504 (A) or SOX6-628 (B) was added. (A) Marcus, CIR-A2402 (SOX6-504), CIR-A2402 (CMV), CIR-A2402 or (B) Marcus, CIR-A2402 ( It is the graph which showed the specific solubility of the target cell calculated when coculturing SOX6-628), CIR-A2402 (CMV), and CIR-A2402 as a target cell. In Examples using the present invention, the proliferation ability of CTL (SOX6-504M) derived from healthy human peripheral blood of HLA-A2402 was measured over time (7th day, 14th day, 21st day, 28th day). It is a graph showing the result measured as a change of cell number. In Examples using the present invention, CTL (SOX6-504M) and CTL (SOX6-504) derived from HLA-A2402 healthy human peripheral blood were used as effector cells, and CIR-A2402, CIR-A2402-SOX6, CIR-A0201 -It is the graph which showed the specific solubility of the target cell calculated when co-cultivating SOX6, Marcus, SF-126, KNS-42, PC9, and HepG2 as a target cell. The specific solubility of the target cells was measured on the 28th day after the start of the experiment. In Examples using the present invention, CTL (SOX6-504M) and CTL (SOX6-504) derived from HLA-A2402 healthy human peripheral blood were used as effector cells, and CIR-A2402, CIR-A2402-SOX6, Marcus, SF It is the graph which showed the specific solubility of the target cell calculated when -126 was co-cultured as a target cell. The specific solubility of the target cells was measured on the 22nd day after the start of the experiment. It is the figure which represented the graph of FIG. 9 as a bar graph which compared the specific solubility with respect to the same target cell. In Examples using the present invention, CTL (SOX6-504M) and CTL (SOX6-504) derived from peripheral blood of HLA-A2402 glioma patients were used as effector cells, and CIR-A2402, CIR-A2402-SOX6, Marcus, SF It is the graph which showed the specific solubility of the target cell calculated when -126 was co-cultured as a target cell. The specific solubility of the target cells was measured on the 22nd day after the start of the experiment. In Examples using the present invention, calculation is performed when CTL (SOX6-504M) is an effector cell and Marcus, CIR-A2402 (SOX6-504), CIR-A2402 (CMV), and CIR-A2402 are co-cultured as target cells. It is the graph which showed the specific solubility of the target cell.

Claims (16)

  A peptide having the sequence of SEQ ID NO: 10.   An antigen-presenting cell presenting a peptide having the sequence of SEQ ID NO: 10.   The antigen-presenting cell according to claim 2, which is a dendritic cell.   A T cell that is induced by the antigen-presenting cell according to claim 2 and recognizes a cell that expresses SOX6.   The T cell according to claim 4, which is a cytotoxic T cell.   The T cell according to claim 4, wherein the cell expressing SOX6 is a glioma.   5. The T cell according to claim 4, wherein the cell expressing SOX6 is a liver cancer cell, lung cancer cell, pancreatic cancer cell, esophageal cancer cell, melanoma, prostate cancer cell, breast cancer cell, renal cancer cell, or leukemia cell.   The T cell according to claim 4, wherein the cell expressing SOX6 is a liver cancer cell, a lung cancer cell, or a pancreatic cancer cell.   A cancer vaccine comprising a peptide having the sequence of SEQ ID NO: 10 and one or more peptides selected from peptides having the sequences of SEQ ID NOs: 1 to 3.   A cancer vaccine comprising the antigen-presenting cell according to claim 2 or 3.   The cancer vaccine containing the T cell in any one of Claims 4-8.   The cancer vaccine according to any one of claims 9 to 11, which is a cancer vaccine against cancer cells expressing SOX6.   The cancer vaccine according to any one of claims 9 to 11, which is a cancer vaccine against glioma.   The cancer vaccine according to any one of claims 9 to 11, which is a cancer vaccine against liver cancer, lung cancer, pancreatic cancer, esophageal cancer, melanoma, prostate cancer, breast cancer, kidney cancer, or leukemia.   The cancer vaccine according to any one of claims 9 to 11, which is a cancer vaccine for liver cancer, lung cancer, or pancreatic cancer. A method for treating a tumor using the cancer vaccine according to any one of claims 9 to 15 against vertebrates other than humans.