JP2021013301A - Hla-a24-restricted epitope peptide derived from methothelin - Google Patents

Abstract

To provide an HLA-A24-restricted epitope peptide derived from methothelin.SOLUTION: Provided are a peptide comprising an amino acid sequence AFYPGYLCSL (SEQ ID NO: 15), a polynucleotide encoding the peptide represented by SEQ ID NO: 15, and an expression vector containing the polynucleotide.SELECTED DRAWING: None

Description

The present application relates to a mesoterin-derived HLA-A24 restrictive epitope peptide. The present application also relates to a cancer vaccine composition containing the peptide of the present application, a method for inducing antigen-presenting cells using the peptide of the present application, and a method for inducing cytotoxic T lymphocytes using the peptide of the present application.

Mesothelin is a glycoprotein expressed on the cell membrane surface of the mesothelial inner layer of the pleura, peritoneum, and pericardial cavity in normal tissues, and is thought to be involved in cell-cell adhesion and signal transduction. In addition, it has been reported that it is overexpressed in malignant tumors such as pancreatic cancer, ovarian cancer, malignant mesothelioma, breast cancer, and lung adenocarcinoma (Non-Patent Documents 1 to 5). Clinical trials with multiple antibody drugs targeting mesothelin have been conducted for various cancers such as mesothelioma, ovarian cancer, pancreatic cancer, lung adenocarcinoma, lung cancer, advanced solid tumor, and pleural mesothelioma. (Non-Patent Document 1).

The present inventors have previously reported that mesothelin plays an important role in the infiltration process of pancreatic ductal adenocarcinoma (PDAC), and that the expression of mesoterin is a poor prognosis factor in patients with pancreatic ductal adenocarcinoma. (Non-Patent Document 2).

As mentioned above, mesothelin is a very attractive target for cancer treatment. Currently, methods of administering tumor-specific cancer antigens, cancer antigen-presenting cells, or cancer antigen-reactive cytotoxic T cells to tumor patients have been developed for the treatment of cancer. Epitope peptides that recognize mesothelin in an HLA-A24 restrictive genotype, which is common in Japanese, are expected to be useful in such therapeutic methods.

Kelly et al., Mol. Cancer Ther. (2012) 11 (3): 517-525 Shimizu et al., Cancer Sci. (2012) 103: 739-746 Tang et al., Anticancer Agents Med. Chem. (2013) 13 (2): 276-280 Madeira et al., An. Acad.Bras. Cienc. (2016) 88 (2): 923-932 Einama et al., World J. Gastrointest. Pathophysiol. (2016) 7 (2): 218-222

An object of the present application is to provide an HLA-A24 restrictive epitope peptide derived from mesothelin. The present application also includes a cancer vaccine composition containing the peptide, a method for producing an antigen-presenting cell that presents the peptide together with HLA-A24, and induction of cytotoxic T cells that recognize the peptide binding to HLA-A24. The purpose is to provide a method.

We have found that the human mesothelin-derived peptide AFYPGYLCSL (SEQ ID NO: 15) induces peptide-specific cytotoxic T cells (CTL) in an HLA-A24 restriction, completing the present invention.

That is, the present application provides a peptide having the amino acid sequence shown in SEQ ID NO: 15. The present application also provides a polynucleotide encoding the peptide set forth in SEQ ID NO: 15, and an expression vector containing the polynucleotide.

The present application also provides a cancer vaccine composition containing the peptide of SEQ ID NO: 15, particularly a cancer vaccine composition for patients having the genotype of HLA-A24.

The present application also provides a method for inducing an antigen-presenting cell that presents the peptide of SEQ ID NO: 15 together with HLA-A24, which comprises expressing the peptide of SEQ ID NO: 15 in HLA-A24-positive cells capable of presenting an antigen.

The present application further comprises culturing HLA-A24 positive peripheral blood mononuclear cells in the presence of a peptide consisting of the amino acid of SEQ ID NO: 15. Cytotoxic T cells that recognize the peptide of SEQ ID NO: 15 in an HLA-A24 constraint. Provides a method of inducing.

The peptides, vectors, antigen-presenting cells and cytotoxic T cells provided by the present application are useful in the treatment of mesothelin-expressing cancer in patients with the HLA-A24 genotype. HLA-A24 is a genotype that accounts for about 60% of Japanese people, and is applied to the treatment of cancer in many Japanese people.

A photograph showing the results of examining the peptide-specific γIFN-producing ability of a part of the CTLs induced using a total of 19 peptides by the ELISPOT assay. Results of examining the peptide-specific γIFN-producing ability of cells obtained by proliferating two oligoclones of CTL derived using the peptide of # 37 (SEQ ID NO: 15) by Rapid Expansion Method (REM method) by ELISPOT assay. Photograph showing. The figure which shows the FACS analysis result of the cell obtained by proliferating the CTL induced by the peptide of # 37 (SEQ ID NO: 15) by the REM method. The graph which shows the result of having examined the cytotoxic activity with respect to the human lymphoblastoma T2A24 strain (HLA-A0201, HLA-A2402) pulsed with the peptide of CTL induced by the peptide of # 37 (SEQ ID NO: 15). The graph which shows the result of having examined the γIFN-producing ability of CTL induced by the peptide of # 37 (SEQ ID NO: 15) for a plurality of pancreatic cancers by an ELISA assay. A photograph showing the results of cloning an oligoclone of CTL derived with the peptide of # 37 (SEQ ID NO: 15) by the limiting dilution method and examining the peptide-specific γIFN-inducing activity of each clone by the ELISPOT assay. The photograph which shows the result of having investigated the peptide-specific γIFN-inducing activity of the clone which each clone of FIG. 6 was grown by the REM method. The photograph which shows the result of having investigated the peptide-specific γIFN-inducing activity of the clone which each clone of FIG. 6 was grown by the REM method.

The present invention provides a peptide consisting of AFYPGYLCSL (SEQ ID NO: 15). The peptide of SEQ ID NO: 15 is an HLA-A24 restrictive mesothelin epitope peptide. That is, when administered to an antigen-presenting cell having HLA-A24, it binds to HLA-A24, which is an HLA class I molecule, is presented on the cell surface, and is recognized by cytotoxic T cells, thereby causing specific cytotoxicity. T cells can be induced efficiently. In addition, cytotoxic T cells established by stimulation with this peptide efficiently recognize tumor cells expressing mesothelin. Therefore, the peptide of SEQ ID NO: 15, the antigen-presenting cell presenting the peptide on the cell surface, and the cytotoxic T cell established by stimulation of the antigen-presenting cell can produce mesothelin in a patient having the HLA-A24 inheritance type. It is useful for the treatment of developing cancer.

As used herein and claimed, "treatment of cancer" can include both treatment and prevention of cancer, preventing the onset of the condition, completely curing the condition, and completely curing the condition. Even if it is not done, it may include suppressing the progress and / or exacerbation of the symptom, delaying the progression of the pathological condition, or improving a part or all of the pathological condition and leading to a cure. Mesoterin is particularly known to be involved in infiltration and migration of cancer cells, and the peptides of the present application are useful for suppressing infiltration, migration and metastasis of cancers expressing mesothelin.

The cancer to be treated with the peptides of the present application is a cancer expressing mesothelin. Cancers that express mesothelin include pancreatic cancer, ovarian cancer, malignant mesoderma, breast cancer, lung adenocarcinoma, esophageal cancer, gastric cancer, liver cancer, colon cancer, bile duct cancer, and uterine cancer. , Head and neck cancer, cartinoid, renal cancer, bladder cancer, prostate cancer, lung cancer and the like are known. The peptides of the present application are particularly useful in the treatment of pancreatic cancer, especially pancreatic duct cancer.

In the specification and claims, when a cell or individual is "HLA-A24 positive" or "has an HLA-A24 genotype", it means that at least one of the alleles is expressed. To do. The type of HLA possessed by the subject can be identified by a routine HLA test.

The peptides of the present application can be produced by ordinary peptide synthesis. As such a method, for example, Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Development of Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991 (the above-mentioned documents are included in the present specification by reference), and the like. Alternatively, it may be a recombinant peptide produced using a gene recombination technique.

The present application provides a cancer vaccine composition for the treatment of a mesothelin-expressing cancer in a patient with HLA-A24, which contains the peptide of SEQ ID NO: 15. In the present application, the "cancer vaccine composition" means a drug which is inoculated or administered to animals including humans and used for the treatment of cancer.

The cancer vaccine composition of the present invention is administered to a patient by, for example, intradermal administration, intralymphatic administration, intracancer tissue administration or subcutaneous administration. The agents of the present invention can include pharmaceutically acceptable salts, carriers and the like to suit such administration. Examples of the salt include alkali metal bicarbonates such as sodium chloride and sodium hydrogen carbonate. Preferably, the drug of the present invention is administered by dissolving it in water or the like so as to be isotonic with plasma. Examples of the carrier include cellulose, polymerized amino acids, albumin, and the like, and if necessary, a carrier to which the peptide used in the present invention is bound can also be used.

The cancer vaccine composition of the present invention may be a liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, or the like. In addition, polysaccharides such as incomplete Freund's adjuvant (for example, ISA-51, etc., SEPPIC) and Purulan, which are conventionally known to be used for vaccine administration, and complete Freund, so that an immune response can be effectively established. It can also be administered together with an adjuvant, BCG, Alam, GM-CSF, IL-2, CpG or the like having an immunopotentiating effect.

The dose of the cancer vaccine composition of the present invention is appropriately adjusted according to the state of the disease, the age of each patient, the body weight, etc., but the amount of peptide in the drug in one administration is usually 0.0001 mg or more. It is 1000 mg, preferably 0.001 mg to 100 mg, more preferably 0.01 mg to 10 mg, even more preferably 0.1 to 5 mg or 0.5 to 3 mg. It is preferable to administer this repeatedly once every few days, weeks, or months.

Vectors The present invention also provides a polynucleotide encoding a peptide of the invention and an expression vector comprising said polynucleotide. When introduced into an antigen-presenting cell, an expression vector containing the polynucleotide of the present invention expresses the peptide of the present invention and causes them to be presented on the cell surface as a complex with an HLA molecule. The antigen-presenting cells can efficiently proliferate CTLs that damage mesothelin-expressing cancer cells.

Expression vectors incorporating the polynucleotide of the present invention include various plasmids and viral vectors, such as adenovirus, adeno-related virus, retrovirus, lentivirus, and vaccinia virus (Liu M, Acres B, Balloul JM, Bizouarne N, Bizouarne N, etc.). Paul S, Slos P, Squiban P. Gene-based vaccines and immunotherapeutics. Proc Natl Acad Sci USA 101 Suppl, 14567-71, 2004). Vector preparation methods are well known in the art (Molecular Cloning: A laboraroy manual, 2nd edn. New York, Cold Spring Harbor Laboratory).

The vector of the present invention can be administered to a patient to express the peptide of the present invention in cells in the patient's body. Alternatively, the vector of the present invention may be introduced into dendritic cells derived from the patient outside the patient's body, and the cells expressing the peptide of the present invention may be returned to the patient. These methods are well known in the art (eg, Hrouda D, Dalgleish AG. Gene therapy for prostate cancer. Gene Ther 3: 845-52, 1996).

When the vector of the present invention is administered to a patient, the dose varies depending on the state of the disease, the age of each patient, the body weight, etc., and for example, the amount of DNA is 0.1 μg to 100 mg, preferably 1 μg to 50 mg. Examples of the administration method include intravenous injection, subcutaneous administration, intradermal administration, intracancerous administration and the like.

[Antigen-presenting cells]
The peptides of the invention can also be used to prepare antigen-presenting cells that induce cancer-reactive cytotoxic T cells (CTLs). That is, the present invention provides a method for inducing an antigen-presenting cell that presents the peptide of SEQ ID NO: 15 together with HLA-A24, which comprises expressing the peptide of SEQ ID NO: 15 in HLA-A24-positive cells having an antigen-presenting ability.

The preparation of antigen-presenting cells is carried out, for example, by culturing HLA-A24-positive cells having an antigen-presenting ability together with the peptide of SEQ ID NO: 15 and causing the peptide to bind and present to an HLA molecule. Alternatively, a vector capable of expressing the peptide of SEQ ID NO: 15 may be introduced into HLA-A24-positive cells having an antigen-presenting ability and expressed. Examples of the antigen-presenting cell include dendritic cells, macrophages, and B cells, but dendritic cells are preferable in consideration of high antigen-presenting ability and the like. Antigen-presenting cells are preferably isolated from the individual to be treated, but may be isolated from other individuals. Alternatively, it may be derived from pluripotent stem cells such as ES cells and iPS cells.

Patient-derived dendritic cells can be obtained, for example, by separating CD8 ^- T cells from peripheral blood mononuclear cells (PBMC) collected from the patient and culturing the cells in the presence of IL-4 and GM-CSF. Be done. The antigen-presenting cells prepared by the above method can induce a CTL that specifically recognizes a complex of a peptide and an HLA molecule presented on the surface of a cancer cell. CTL can be induced in vitro by culturing such antigen-presenting cells together with PBMC-derived CD8 ⁺ T cells collected from a patient. In addition, when such antigen-presenting cells are administered to a patient having a cancer expressing mesothelin, the induction of cancer-reactive CTL can be promoted in the patient's body. Therefore, the antigen-presenting cells prepared using the peptide of SEQ ID NO: 15 can be used as a drug for treating cancer.

The administration site of the antigen-presenting cells may be intradermal administration, subcutaneous administration, intravenous administration, lymph node administration, intraperitoneal administration, or the like, and is not particularly limited. However, considering that physiological anti-cancer immune responses including antigen presentation of physiological dendritic cells occur in the cancer tissue and in the vicinity of the regional lymph node of the dendritic cell administration site, the inside of the cancer tissue Alternatively, direct administration into the lymph nodes is preferable.

[Cancer antigen-reactive cytotoxic T cells]
The present application also provides a method for inducing cytotoxic T cells (CTL) that specifically recognize HLA-A24 positive and mesothelin-expressing cancer in vitro.

The peptides of the invention can also be used to induce cancer-reactive CTLs in vitro. That is, the present invention provides a method for inducing cancer-reactive CTL, which comprises contacting a PBMC collected from a cancer patient or a healthy person with a peptide of the present invention. Induction of CTL is performed, for example, by culturing PBMC collected from a cancer patient or a healthy person in vitro in the presence of the peptide of the present invention. Alternatively, an antigen-presenting cell that presents the peptide of SEQ ID NO: 15 derived by the above method together with HLA-A24 may be co-cultured with a CD8 ⁺ fraction of PBMC. The resulting CTL is useful for adoptive immunotherapy that damages mesothelin-expressing cancer cells.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any sense.

Selection of Mesoterin -derived, HLA-A24-binding epitope peptide (1) Prediction of Mesoterin-derived HLA-A24-binding peptide For 9 mer and 10 mer peptide fragments of the Mesoterin amino acid sequence, use BIMAS software to determine the binding affinity for HLA-A24. Calculated using. From each peptide fragment, 10 and 9 peptides, respectively, having high affinity were selected, synthesized by a conventional method, and subjected to the following tests. The selected peptides are shown in Table 1.

The HLA-A2402 binding HIV epitope peptide (RYLKDQQLL) was used as a negative control and the HLA-A24 binding CMV peptide (QYDPVAALF) was used as a positive control. The peptide was dissolved in DMSO at a concentration of 20 mg / ml and stored at -80 ° C.

(2) Cell line For the human lymphoblastoma T2A24 strain (HLA-A0201, HLA-A2402), cells provided by Dr. Kuzushima of the Aichi Cancer Center were used. Burkitt lymphoma Jiyoye (HLA-A32, B17, Bw37) and EB-3 (HLA-A3, Aw32, Cw2), pancreatic cancer PANC-1, pancreatic cancer ASPC1 cell lines are from the American Type Culture Collection (Virginia, USA) Purchased from Manassas, VA). The T2A24 cell line contains RPMI 1640, 10% heat-inactivated FBS (FBS; Biowest, France) and 0.8 mg / ml Geneticin (R) Selective Antibiotic (Thermo Fisher Scientific, Waltham, MA) -containing HEPES medium (Thermo Fisher Scientific, Waltham, It was maintained at MA).

Jiyoye and EB-3 cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin streptomycin solution ((Thermo Fisher Scientific, Waltham, MA). KP2, KP3, KP4, SUIT2 cells were Purchased from JCRB Cell Bank, National Institute of Pharmaceutical Sciences, Health and Nutrition.

(3) In vitro peptide-specific CTL induction CD8 ⁺ T cells and dendritic cells (DC) were prepared from peripheral blood mononuclear cells (PBMC) of healthy individuals having the genotype of HLA-A2402. PBMC the Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) by isolated, CD8 ⁺ T cell isolation Kittohito (Miltenyi Biotech Co., Ltd., Japan) CD8 ⁺ T cell population and CD8 by ^- was separated into cell populations. Monocyte-rich CD8 ^- cell populations were cultured in AIM-V medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS). After overnight culture, non-adherent cells were removed to remove 1000 U / ml granulocyte macrophage colony stimulator (GM-CSF; R & D Systems, Minneapolis, Minnesota, USA) and 1000 U / ml interleukin-4 (IL-4). ) (R & D Systems, Minneapolis, Minnesota, USA) was added to the culture medium to induce monocyte-derived dendritic cells. On the 4th day, 0.1 KE / ml of OK-432 (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) was added to the culture medium to induce the maturation of DC. On day 7, DC cells were pulsed in 3 μg / ml β2-microglobulin (Sigma-Aldrich, St. Louis, MO) -containing AIM-V medium containing 20 μg / ml of each synthetic peptide (37 ° C.). 3 hours).

Peptide-pulsed DC was incubated at 37 ° C. for 30 minutes in medium containing 30 μg / ml mitomycin C (MMC) (Kyowa Hakko Kirin Co., Ltd., Tokyo). After washing away the residual peptides and MMC, DC and autologous CD8 ⁺ T cells were co-cultured on a 48-well plate (Corning Inc., Corning, NY). On day 0, each well contained 1.5 × 10 ⁴ peptide pulse DCs and 3 × 10 ⁵ CD8 ⁺ T cells with 2% AS supplemented with 10 ng / ml IL-7 (R & D Systems). Culturing was started in AIM-V medium. Two days later, human IL-2 (Pepro Tech, Rocky Hill, NJ, USA) was added to the culture (final concentration 20 IU / ml). On days 14 and 21, T cells were further stimulated with freshly prepared peptide pulse autologous DCs each time. Cells were harvested on day 28 and the induction of peptide-specific CTL was assessed by IFN-γ enzyme-binding immune spot (ELISPOT) assay under stimulation with peptide pulsed T2A24 cells.

(4) IFN-γ enzyme-binding immunospot (ELISPOT) assay Peptide-specific CTL response is examined by ELISPOT assay using the IFN-γ ELISPOT kit and AEC substrate set (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. It was. 20 μg / ml of each peptide was pulsed to T2A24 cells at 37 ° C. for 20 hours to wash away residual peptides that did not bind to the cells to obtain stimulated cells. After removing 500 μl of the supernatant from each well in which the peptide-specific CTL was induced, 100 μl of the cell suspension was collected from each well and co-cultured with stimulating cells (1 × 10 ⁴ cells) on an ELISPOT plate (1 × 10 ⁴ cells). 20 hours at 37 ° C). For comparison, the cells were co-cultured with stimulated cells that did not undergo peptide pulse. The obtained spots were measured using an ELIPHOTO Counter (Minerva Tech KK Japan). The results are shown in FIG.

When 100 spots or more were confirmed per well and the number obtained by subtracting the number of spots of peptide (−) wells from the spots of peptide (+) wells was 50 or more, it was determined that “there was peptide-specific CTL activity”.

As shown in FIG. 1, peptide # 37 (SEQ ID NO: 15) was determined to have the strongest peptide-specific CTL activity. In the following tests, peptide # 37 (SEQ ID NO: 15) was used.

Proliferation and activity confirmation of cytotoxic T cells induced by the peptide of SEQ ID NO: 15 (1) Proliferation culture of cytotoxic T cells Two of the CTL cells induced by the peptide of SEQ ID NO: 15 were confirmed to be ELISPOT positive. The cells (# 37-E2 and # 37-E4) obtained from the wells were used in the previously reported REM method (Rapid Expansion Peptide) (Wada S, Tsunoda T, Baba T, Primus FJ, Kuwano H, Shibuya M, Tahara H. Rationale). For antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2. Cancer Res. 4939-46. 2005). On day 0, the remaining CTL cell suspension used in the ELISPOT assay was subjected to MMC-treated (30 μg / ml, 37 ° C. for 30 minutes) with 40 ng / ml antibodies along with 5 × 10 ⁶ EB-3 cells. Culturing was initiated in 25 ml of 5% self-serum-added AIM-V medium containing a CD3 monoclonal antibody (BD Pharmingen, San Diego, CA). 5% autologous serum-containing AIM- with IL-2 added on day 1 (final concentration 120 IU / ml) and medium on days 5, 8 and 11 with 30 IU / ml IL-2 added. It was replaced with V medium. On day 14, cell CTL activity was examined by IFN-γ ELISPOT assay.

The CTLs grown by the REM method were further grown twice by the REM method, and the activity of the grown CTL cells obtained by each growth procedure was examined by the IFN-γELISPOT assay. The results are shown in FIG. In addition, FIG. 3 shows the results of FACS analysis of cells grown by the REM method a total of 3 times.

As shown in FIG. 2, peptide-specific CTL activity was maintained even after repeated culture by the REM method. Further, from FIG. 3, it was confirmed that the obtained cells were CD8 single positive cells.

(2) Cytotoxicity assay The cytotoxic activity of peptide-specific CTL was examined by a cytotoxicity assay based on fluorescence intensity.

Fluorescence intensity-based cytotoxicity testing was performed using the DELFIA EuTDA cytotoxic activity assay reagent. Peptide-pulsed and non-peptide-pulsed T2A24 cells were used as target cells. Each cell was labeled with DELFIA BATDA reagent at 37 ° C. for 30 minutes as instructed. BATDA-labeled target cells were then incubated with the peptide-specific CTLs derived above for 4 hours. After incubation, supernatants were collected and measured by time-resolved fluorescence. In addition, 35% hydrochloric acid was added to the cells and the maximum release was measured. Cytotoxicity (%) calculated by the following formula = [(release in test group-spontaneous emission) / (maximum release-spontaneous emission)] x 100

The results are shown in FIG. It was confirmed that both E2 and E3 CTLs show peptide-specific cytotoxic activity against the peptide of SEQ ID NO: 15 (# 37) in T2A24 cells expressing HLA-A2402.

Mesoterin and HLA-A2402 specific activity of cytotoxic T cells induced by the peptide of SEQ ID NO: 15 (1) IFN-γ assay by ELISA As target cells, pancreatic cancer cell lines KP2, PANC1, ASPC1, KP4 , SUIT2 and KP3, respectively, and the IFNγ-producing ability of the # 37-E4 cell group was examined. Using 96-well round bottom plates, CTL cells were co-cultured with 5 × 10 ⁴ target cells for 20 hours in 200 μl of 5% self-serum-added AIM-V medium for each well. After culturing, cell-free supernatants are collected and IFN-γ production is measured using human IFN-γ set (BD Biosciences) and BD OptEIA ™ reagent set B (BD Biosciences) as described in the instructions. did. The presence or absence of expression of mesothelin and HLA-A2402 in each cell line is summarized below:

The results are shown in Fig. 5. IFN-γ production was observed when co-cultured with pancreatic cancer cell lines (KP2, SUIT2 and KP3) expressing both mesothelin and HLA-A2402, but not expressing either or both antigens. When co-cultured with a cell line, almost no IFN-γ was produced. From this result, it was confirmed that the peptide of SEQ ID NO: 15 induces CTL showing a specific reaction to cancer cells expressing mesothelin and HLA-A2402.

Establishment of Peptide-Specific CTL Clone A peptide-specific CTL clone was established by the limit dilution method. The # 37 peptide-specific CTL (E4), which was repeated twice in REM, was diluted in a 96-well round bottom plate (Corning, Inc.) to 0.3, 1 and 3 cells / well, and treated with MMC ( It was cultured with Jiyoye and EB-3 cells 1 × 10 ⁴ cells / well at 37 ° C. (30 μg / ml). As the medium on day 0, AIM-V medium containing 5% self-serum containing 30 ng / ml of anti-CD3 monoclonal antibody and 125 IU / ml of IL-2 was used. On day 10, IL-2 was added to each well (125 UI / ml). On day 14, an IFN-γELISPOT assay was performed to measure the CTL activity of each clone. The results are shown in FIG.

In addition, each of the obtained clones was grown by the REM method and then subjected to the IFN-γELISPOT assay. The results are shown in FIGS. 7 and 8.

Cloning resulted in a CTL cell clone having a peptide-specific IFN-γ producing ability, and proliferation by the REM method resulted in proliferation of the clone while maintaining CTL activity.

Claims (10)

A peptide consisting of the amino acid sequence of AFYPGYLCSL (SEQ ID NO: 15). A polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 15. An expression vector containing the polynucleotide of claim 2. A cancer vaccine composition for the treatment of mesothelin-expressing cancer in patients with the HLA-A24 genotype, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 15. The composition according to claim 4, which suppresses cancer infiltration, migration and / or metastasis. Cancers are pancreatic cancer, ovarian cancer, malignant mesoderma, breast cancer, lung adenocarcinoma, esophageal cancer, gastric cancer, liver cancer, colon cancer, bile duct cancer, uterine cancer, head and neck cancer, The composition according to claim 4 or 5, selected from the group consisting of cartinoids, renal cancer, bladder cancer, prostate cancer and lung cancer. A method for inducing an antigen-presenting cell that presents the peptide of SEQ ID NO: 15 together with HLA-A24, which comprises expressing the peptide of SEQ ID NO: 15 in HLA-A24-positive cells having an antigen-presenting ability. The method according to claim 7, wherein the cell having an antigen-presenting ability is a dendritic cell. A method for inducing cytotoxic T cells that recognize the peptide of SEQ ID NO: 15 in an HLA-A24-restricted manner, which comprises culturing HLA-A24-positive peripheral blood mononuclear cells in the presence of a peptide consisting of the amino acid of SEQ ID NO: 15. .. Recognizes the peptide of SEQ ID NO: 15 in an HLA-A24 constraint, which comprises co-culturing CD8-positive cells with antigen-presenting cells that present the peptide of SEQ ID NO: 15 according to claim 7 or 8 together with HLA-A24. A method for inducing cytotoxic T cells.