KR20110134482A - Hla-a24-binding cancer antigen peptide derived from sox2 - Google Patents

Abstract

The present invention provides a peptide capable of inducing cytotoxic T cells targeting cancer cells, wherein the peptide comprises an amino acid sequence derived from a polypeptide encoded by the Sox2 gene, or one or more amino acid sequences thereof. A few amino acids contain an amino acid sequence that is deleted, substituted or added, and also contains the peptide, the CTL derived by the peptide, the peptide and / or the CTL, antigenically presented by HLA-A24. It relates to a pharmaceutical composition. The invention also relates to the use for the CTL induction of the peptide.

Description

HLA-A24-binding cancer antigen peptide derived from SO2 {HLA-A24-BINDING CANCER ANTIGEN PEPTIDE DERIVED FROM SOX2}

The present invention relates to peptides capable of inducing cytotoxic T cells (CTLs) targeting cancer cells.

The present invention also relates to a cancer vaccine and an anticancer agent comprising the peptide.

The present invention further relates to the use of the peptide for inducing CTLs targeting cancer cells, and to the obtained CTL and an anticancer agent comprising the CTL.

Recent advances in immunology and molecular biology have greatly influenced the progress of tumor immunity. When an influenza virus infection occurs in humans, the idea that immunity is established against the infection and released from the infection can be explained by the following cellular immunity. Epithelial cells infected with influenza virus present 9-10 peptides from the viral genome on the major histocompatibility antigen complex HLA molecules on their cell surface. Infected cells presenting this HLA-virus peptide complex cause an intense allogeneic response, which is specifically recognized by CD8 positive CTLs present in peripheral blood and actively excluded. Such a mechanism of cellular immunity is understood to work in the same way for cancer cells caused by the tumor cells of their own. This has been demonstrated by the isolation of tumor antigen MAGE genes from malignant melanoma by Thierry Boon et al. (Van der Bruggen et al., Science, 254, 1643-1647 (1991)).

As a method for identifying cancer antigens recognized by T cells, a method of screening a cDNA library derived from human cancer using T cells has already been developed, and the MAGE gene described above has been isolated using this method. Subsequently, a plurality of cancer-derived cancer antigen peptides present on the surface of cancer cells including malignant melanoma and recognized as T-cells have been identified, and clinical trials using several of them have been initiated. have. For example, the NY-ESO-1 molecule identified from esophageal cancer as a molecule recognized by an antibody present in the serum of cancer patients is recognized that the synthetic peptide has induction ability of CTL (Chen, YT. Et al. , Proc. Natl. Acad. USA, 94 , 1914-1918 (1997) and Jager, E. et al., J. Exp. Med., 187 , 265-270 (1998).

However, in epithelial cancers such as colorectal cancer, stomach cancer, breast cancer, lung cancer, and bladder cancer, which occupy most cancers, cancer antigens are hardly identified, and immunotherapy targeting cancer antigens is not established.

Sex determining region Y-box2 (Sox2) gene, which is expressed in the central nerve of the natal period and is known as a gene involved in self-replication of neural stem cells, is known to be expressed in many malignant glioma. have. The publication of M. Schmitz et al. Discloses that it is possible to artificially cause CTL induction by using HLA-A2 binding epitope of Sox2 and to eliminate glioma (Non-Patent Document 8).

In recent studies, many Sox2 genes are also expressed in cancer tissues including cancer stem cells which are considered to be a major cause of cancer recurrence or metastasis (Non Patent Literatures 1 to 7).

[Non-Patent Document 1] Nakatsugawa Munehide et al., “SOX2 as a Cancer Stem Cell Antigen Molecule”, Abstract of the 12th Foundation Cancer Immunity Meeting, p. 39 [Non-Patent Document 2] Toshihiko, Torikoshi et al., "Survivin2B Peptide Vaccine Clinical Trial: From Bed to Bench," Journal of the Korean Society for Clinical Immunology Vol. 31, No. 4, p. 244 [Non-Patent Document 3] Akari Takahashi et al., Cancer Stem Cell Antigen SOX2 of Breast Cancer and Lung Cancer, 88th Hokkaido Medical Conference Program / Abstract, page 17 [Non-Patent Document 4] Shoji Sato, `` Human Cancer Stem Cell Antigen Analysis and Immune Response, '' 67th Annual Meeting of the Japan Cancer Society, p. 82 [Non-Patent Document 5] Asanuma Hiroko et al., "Basaloid breast cancer expresses stem cell markers with a high frequency," article at the 67th meeting of the Japanese Society of Cancer Studies, page 177. [Non-Patent Document 6] Aka Takahashi et al., "Searching for Cancer Stem Cell Antigens in Lung Cancer, Breast Cancer, and Sarcoma," Article 67th Japanese Society of Cancer Society, p. 443 [Non-Patent Document 7] Yoshihiko Hirohashi et al., "Immunologic Evaluation of Lung Cancer Stem Cells", Article 67th Japanese Society of Cancer Studies Academic Conference, p. 440 [Non-Patent Document 8] M. Schmitz et al., British Journal of Cancer (2007) 96, 1293-1301.

An object of the present invention is to provide a cancer vaccine and an anticancer agent which can be used for immunotherapy of cancer.

The inventors noted that mainly CTLs, in particular CD8 (+) CTL, are responsible for immunological human cancer rejection. CD8 (+) CTLs are activated by recognizing a complex comprising a major histocompatibility antigen complex (HLA in humans) on cancer cells and a cancer antigen peptide presented on the HLA. The activated CTL recognizes and attacks cancer cells through a T cell antigen receptor on its cell surface. Therefore, once a cancer antigen peptide is identified, it can be used as a cancer vaccine and an anticancer agent to efficiently induce CTL to prevent and treat cancer.

SOX2 is a gene transcription regulator, and expression level is particularly high in cancer stem cells having stem cell-like traits among cancer cells. SOX2 is expressed in many cancers such as lung cancer and kidney cancer, but expression in normal tissues is limited to fetal stem cells and neural stem cells.

The present inventors have studied the cancer antigenicity, that is, the CTL inducibility of various SOX2 derived peptides, and there is an HLA-A24 binding motif in an amino acid sequence including 317 encoded by the SOX2 gene. It was found that certain peptides could induce CTLs. This finding led to the completion of the present invention.

In other words, the present invention provides a peptide capable of inducing cytotoxic T cells targeting cancer cells, wherein the peptide comprises an amino acid sequence derived from a polypeptide encoded by the Sox2 gene or in the amino acid sequence. The peptide relates to an amino acid sequence in which dogs or several amino acids are deleted, substituted or added and which is antigenically presented by HLA-A24.

The present invention also relates to the above, wherein the peptide includes an amino acid sequence represented by SEQ ID NOs: 1, 4 to 9, and 12 or a sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence. It relates to a peptide.

The present invention also relates to cytotoxic T cells induced by the above peptides.

The present invention also relates to DNA encoding the above peptide.

Moreover, this invention relates to the pharmaceutical composition containing said peptide and / or said cytotoxic T cell, and / or said DNA.

The present invention also relates to the above pharmaceutical composition, wherein the pharmaceutical composition is a cancer vaccine.

The present invention also relates to the above pharmaceutical composition, wherein the pharmaceutical composition is an anticancer agent.

The present invention also relates to the above pharmaceutical composition, wherein the pharmaceutical composition is a DNA vaccine.

The present invention also relates to the use of said peptide for inducing cytotoxic T cells targeting cancer cells.

The modified peptide which modified the peptide of this invention and 1-2 amino acids can induce CTL which injures the cancer cell which is HLA-A24 positive SOX2 positive thought to be showing the peptide of this invention. The expression rate of HLA-A24 is 20-30% for Westerners and 50% or more for Japanese. Accordingly, the cancer antigen peptides of the present invention can be used worldwide as cancer vaccines and anticancer agents useful for various types of SOX2 positive malignancies.

1 shows the results of an HLA-A24 binding assay of a SOX2 polypeptide-derived peptide. Peptides showing MFI at or near the same level as the peptide used as the positive control were identified as HLA-A24 binding peptides.
Fig. 2 shows the results of cytotoxicity analysis of the SOX2_109 peptide. Significantly greater cellular injury has been detected for HIV-derived peptides, which are negative controls.
Fig. 3 shows the results of ELISPOT analysis of the SOX2_109 peptide. The release of IFNγ was not detected in the HIV-derived peptide as a negative control, whereas the release of IFNγ was confirmed in the SOX2_109 peptide.
4 shows the results of ELISPOT analysis of SOX2 derived peptides. In the figure, sur2B represents the survivin2B addition group as a negative control, and (-) represents the group without addition of the peptide. CTLs responding to SOX2_50 and _58 were not induced in any patients, and CTLs responding to SOX2_124 and 216 were derived from four patients.
Fig. 5A is a diagram showing the results of cytotoxicity analysis on T2-A24 cells antigen-presenting SOX2-derived peptides, and b) cells in LHK2 lung cancer cells and normal LHK2 cells forcibly expressing SOX. It is a figure which shows the result of an injury analysis.

Hereinafter, the present invention will be described in detail.

Identification of the peptide of the present invention is carried out in the following steps:

(1) providing a peptide derived from SOX2 polypeptide having a sequence corresponding to the binding motif of HLA-A24 which is a human major histocompatibility antigen complex (MHC) class I,

(2) adding the peptide to antigen-presenting cells expressing HLA-A24 to obtain antigen-presenting cells presenting the peptide by HLA-A24;

(3) stimulating T cells by the antigen presenting cells to induce CTL, and

(4) Process of measuring cancer cell injury ability of induced CTL

It can be performed by the method containing.

Since the peptide of the present invention has a small number of amino acids of 10, it can be synthesized by chemical synthesis of general amino acids such as Fmoc. It can also be synthesized using a commercially available amino acid synthesizer. Moreover, since the peptide of this invention originates in the SOX2 polypeptide expressed in the inside of cancer cells, it is described in Suzuki, K. et al., J. Immunol., 163, 2783-2791 (1999) from cancer cells of cancer patients. According to the method, a cell surface HLA binding peptide can be isolated to obtain a corresponding peptide.

The peptide of the present invention includes those in which one or a plurality of amino acids of the peptide are modified by number. Modified peptides are also capable of inducing CTL induction by HLA-A24, which injures cells which antigen-present a SOX2 polypeptide-derived peptide.

The peptide of the present invention can induce cytotoxic T cells targeting cancer cells. The cells expressing HLA-A24 used for induction may be obtained from cancer patients, or may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.

As the peptide of the present invention, any peptide capable of inducing CTLs that injure cells presented by HLA-A24 as a peptide derived from SOX2 polypeptide may be used, and has a certain degree of binding affinity with HLA-A24. In the above, peptides represented by SEQ ID NOs: 1, 4 to 9, and 11 are preferable.

DNA encoding the peptide of the present invention is also included in the present invention. The DNA encoding the peptide of the present invention may be any one that produces the peptide of the present invention when transcribed or translated, but is preferably the same sequence as the sequence encoding the peptide portion of the present invention among the Sox2 gene sequences. DNA.

When using the peptide or DNA of this invention as a pharmaceutical composition, the peptide or DNA of this invention can be used by itself or with an adjuvant, and can contain a pharmaceutically acceptable carrier suitably. As an adjuvant, an adjuvant aimed at enhancing an immune response, for example, an incomplete (complete) adjuvant of a Freund, an aluminum adjuvant, a viral envelope vector, and the like.

Examples of the pharmaceutically acceptable carrier include PBS, diluents such as distilled water, physiological saline, and the like.

DNA used as a pharmaceutical composition includes not only DNA but also the vector construct which inserted the DNA of this invention. As the vector, any material capable of acting as the pharmaceutical composition of the DNA of the present invention may be used, and a vector suitable for the form and purpose of the pharmaceutical composition can be appropriately employed by known techniques in the art. Examples of the vector include, but are not limited to, a plasmid vector, a sendivirus, a retrovirus, a vaccinia virus, an adenovirus, an adeno-associated virus, a herpes virus, an influenza virus, and the like.

The pharmaceutical composition of the present invention can be used in the form of a cancer vaccine, an anticancer agent or a DNA vaccine. Cancer vaccines, anticancer agents or DNA vaccines of the present invention can be prepared by methods well known in the art such as liquids, emulsions, emulsions, soft capsules, hard capsules, tablets, granules, solids, and the like. You can do that.

Cancer vaccines and anticancer agents of the present invention can be administered orally, parenterally or transdermally, depending on the form of use. For example, intravenous administration, root administration, subcutaneous administration, intradermal administration, etc. are mentioned. The dosage usually depends on the weight of the patient, the nature and condition of the disease, but when used in adults, it is up to 5-10 mg per day. For example, when used by subcutaneous injection in adult cancer patients, it is 0.1-10 mg per week, Preferably it is 100-1,000 micrograms.

In addition, since CTL obtained using the peptide of the present invention targets cancer cells, it can be used as an anticancer agent. In this case, similarly to the anticancer agent containing the peptide of the present invention described above, an appropriately pharmaceutically acceptable carrier can be included and various forms can be taken. The anticancer agent containing the CTL of the present invention can be parenterally administered similarly to a cancer vaccine and an anticancer agent containing the peptide of the present invention.

Cancers to which the cancer vaccines, anticancer agents and DNA vaccines of the present invention can be applied are cancers, such as epithelial cancers, consisting of cancer cells presenting the peptides of the present invention by HLA-A24. Examples of the epithelial cancer include lung cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer and breast cancer.

In addition, the peptides of the present invention can be used to induce CTLs that target cancer cells. Induction can be carried out according to the method described, for example, in Nabeta, Y. et al., Jpn. J. Cancer Res, 91, 616-621 (2000).

Specifically, the following process:

(1) providing a cell expressing HLA-A24,

(2) adding the peptide of the present invention to the cells and presenting on HLA-A24,

(3) stimulating T cells with cells presenting the peptide by HLA-A24 to induce the T cells to cancer cell target CTL

It can be used a method including.

The cells expressing HLA-A24 may be obtained from cancer patients, or may be prepared by introducing a gene encoding HLA-A24 into a non-HLA-A24 expressing cell.

[Example]

The following examples further illustrate the present invention and do not limit the scope of the present invention. As those skilled in the art, various modifications can be made to the embodiments shown in the following Examples without departing from the spirit of the present invention, but such modified embodiments are included in the present invention.

Example  One

Sox2  Of the derived peptide HLA -A24 Peptide Binding Assay

The amino acid sequence of Sox2 is shown by SEQ ID NO. The peptide which binds to HLA-A24 is known that the second amino acid is tyrosine, tryptophan, phenylalanine or methionine, and the amino acid at the C terminus is leucine, isoleucine, tryptophan, phenylalanine or methionine. Of the sequences included in the amino acid sequence of Sox2, 9 to 11 amino acid sequences having this HLA-A24 binding motif were selected, and a total of 13 types of the following peptides were synthesized.

SOX2_1: MYNMMETEL (SEQ ID NO 1)

SOX2_50: VWSRGQRRKM (SEQ ID NO: 2)

SOX2_58: KMAQENPKM (SEQ ID NO: 3)

SOX2_89: PFIDEAKRL (SEQ ID NO: 4)

SOX2_109: KYRPRRKTKTL (SEQ ID NO: 5)

SOX2_119: LMKKDKYTL (SEQ ID NO: 6)

SOX2_124: KYTLPGGLL (SEQ ID NO: 7)

SOX2_165: GWSNGSYSM (SEQ ID NO: 8)

SOX2_170: SYSMMQDQL (SEQ ID NO: 9)

SOX2_196: PMHRYDVSAL (SEQ ID NO: 10)

SOX2_209: SMTSSQTYM (SEQ ID NO: 11)

SOX2_216: YMNGSPTYSM (SEQ ID NO: 12)

SOX2_226: SYSQQGTPGM (SEQ ID NO: 13)

T2-A24 cells are cell lines expressed by introducing the HLA-A2402 gene into human lymphoid subpopulation T2 cells. Low levels of HLA-A24 molecules are expressed on the cell surface of these cells, and can be detected using HLA-A24 specific monoclonal antibodies and flowmeters. Expression levels are quantified as mean fluorescence intensity (MFI). The addition of synthetic peptides in vitro to these cells increases the cell surface HLA-A24 expression level in correlation with binding affinity when the added peptide binds to the HLA-A24 molecule. Using the above experimental system, HLA-A24 binding affinity of the SOX2 derived cancer antigen peptide of the present invention was analyzed.

T2-A24 cells were incubated overnight at 26 ° C. Cells were then washed with PBS, a synthetic peptide derived from SOX2, HIV peptide derived from human immunodeficiency virus as a positive control (SEQ ID NO: 14), EBV peptide derived from Epstein-Barr virus (SEQ ID NO: 15), and As a negative control, SL8 peptide (SEQ ID NO: 17), a peptide derived from egg white albumin, was added and co-cultured at 26 ° C for 3 hours. The temperature was 37 [deg.] C. and co-cultured for an additional 2.5 hours, then centrifuged to remove the supernatant and the cells isolated. HLA-A24 antibody (C7709A2.6) was added to the isolated cells, and allowed to stand at 4 ° C for 1 hour, followed by washing with PBS. Fluorescently labeled anti-mouse IgG + IgM antibody was added as a secondary antibody, left at 4 ° C. for 30 minutes, and then cells were fixed by adding 1% formalin. The fixed cells were measured for FITC fluorescence intensity by a flowmeter (BD FACS Calibur).

The results are shown in FIG. Eight SOX2 peptides with fluorescence intensity equal to or greater than positive control were determined as HLA-A24 binding peptides.

Example  2

CTL  Judo

Peripheral blood mononuclear cells (PBMC) by centrifugation in a Ficoll-Con ray density gradient from 50 ml of peripheral blood of HLA-A24 positive lung cancer patients or HLA-A24 positive healthy normal persons obtained informed concent Was separated and recovered. Then, CD8 MACS beads were used to separate CD8 positive lymphocytes and CD8 negative lymphocytes from PBMC.

In a 96 well plate, 200,000 / well CD8 negative lymphocytes and the HLA-A24 binding SOX2 peptide were respectively mixed, allowed to stand at room temperature for 2 hours, and then irradiated with 100 Gy. Treated cells were co-cultured with 20 U / ml IL-2 (Takeda Yakuhin High School), 100,000 / well CD8 positive lymphocytes, followed by CD8 positive lymphocyte stimulation once a week, and stimulated three times in total for CTL Induced. Peptide specific reactivity of induced CTLs was assessed by cytotoxicity assay or ELISPOT assay.

Example  3

Cell injury analysis

Cr ⁵¹ was added to T2A24, incubated for 1 hour at 37 ° C. in RPMI medium, radiolabelled, and washed four times in RPMI medium. SOX2 peptide or control peptide (HIV peptide) was mixed with Cr-labeled T2A24, and allowed to stand at room temperature for 1 hour. 2,000 / well T2A24 pulsed with SOX2 peptide or control peptide and 14,000 / well CD8 positive lymphocytes derived from Example 2 were co-cultured at 37 ° C. for 4 hours in RPMI medium. The supernatant was then collected and gamma dose was measured by a gamma counter.

In addition, the dose in the case of RPMI medium only and in the case of culturing peptide pulse T2A24 is natural release (SPR), the dose in the case of culturing cell lysate (2% NP40) and peptide pulse T2A24 is the maximum release (MXR). And cytotoxic activity were calculated as (measured value-SPR) / (MXR-SPR) × 100.

As a result, CTLs that specifically interfere with T2A24 pulsed SOX2_109 peptide were detected. The results are shown in FIG. In HIV-derived peptides, which are control peptides, almost no radiation from injured cells was detected, whereas in SOX2_109 peptides, many were detected.

Example  4

ELISPOT  analysis

The experiment was performed using Human IFNγ ELISPOT set (BD). Anti IFNγ antibodies were coated on ELISPOT plates by standing at 4 ° C. overnight. SOX2 peptide or control peptide (HIV peptide) was added to T2A24, respectively, and allowed to stand at room temperature for 1 hour. 50,000 peptide pulsed T2A24 and 10,000 CTLs derived from Example 2 were co-cultured overnight at 37 ° C. in a plate coated with an anti-IFNγ antibody. After washing with 1 × PBS and 0.05% Tween20, a biotin-labeled anti-IFNγ antibody was added and allowed to stand for 2 hours at room temperature. After washing with 1 × PBS and 0.05% of Tween20, HRP-labeled streptavidin was added and allowed to stand at room temperature for 1 hour. After washing with 1 × PBS and 0.05% of Tween20, a coloring reagent was added to determine the number of spots.

The results are shown in FIG. CTLs that specifically reacted against T2A24 pulsed SOX2_109 peptide were detected. In the HIV-derived peptide, which is a control peptide, no release of IFNγ from T cells was detected, but it was confirmed that many IFNγ were released from the SOX2_109 peptide.

Example  5

bracket SOX2  Cytotoxic T Cells of Derived Peptides Induction  compare

Peripheral blood from 6 patients with HLA-A2402 positive lung cancer was used. All patients obtained informed consent. PBMCs were separated by Lymphoprep (registered trademark) (Nycomed, Oslo, Norway) according to the density gradient centrifugation method of the conventional method.

(1) CTL derivation

PBMCs isolated above were seeded to 1 × 10 ⁷ PBMCs / well and cultured in AIM medium (Life Technologies, Inc.) containing 10% pooled human AB serum. cDNA encoding the full length of SOX2 was inserted into pcDNA 3.1 plasmid (Invitrogen), and it was embedded in mannose-coated liposome (OML-SOX2, manufactured by Biomedcore Co., Ltd.). On day 1, OML-SOX2 was pulsed at a concentration of 0.1 μg / mL or 1 μg / mL and incubated for 1 week with the addition of 50 U of IL-2. Subsequently, SOX2-derived peptide specific CTLs were detected by Enzyme-Linked Elisport (hereinafter referred to as "ELISPOT") analysis.

(2) ELISPOT analysis

The specificity of CTLs for the SOX2-derived peptides was determined by IFNγ ELISPOT analysis according to a previously reported method (Tsuruma, T et al., J. Transl. Med., 6:24 (2008)). First, add 5 μg / mL anti-IFNγ capture antibody (Beckton Dickinson Biosciences) in PBS at 100 μL / well to a multi-screen 96 well plate (Millipore, Bedford, Mass.) And overnight at 4 ° C. Aseptically while standing. Plates were washed once with 200 μL / well and blocked for 2 hours at room temperature using 200 μL / well of AIM-V medium with 10% human serum. As antigen-presenting cells, a subcellular C1R-A24 cell of a C1R lymphoma expressing HLA-A24 was pulsed with a survivin2B peptide as 10 μg / mL of each of the above SOX2 peptides and negative control. Thereafter, 5 × 10 ³ CTLs were co-cultured with 5 × 10 ⁴ antigen presenting cells C1R-A24 cells. After incubation for 24 hours at 37 ° C., 5% CO ₂ , wells were washed five times with PBS and incubated with biotinylated anti-human IFN-γ antibody and horseradish peroxidase binding avidin. IFNγ spots formed were counted using KS ELISPOT (Carl Zeiss).

The results are shown in FIG. The numbers in the table mean the measured spot numbers. It was judged positive that the number of spots 1.3 times or more of either of a high value among the negative control sur2B and (-) was measured. Below the table the number of patients positive for each peptide is shown. CTLs that respond to SOX2_50 and _58 are not derived from any patient and are considered to be the least antigenic peptides. In contrast, CTLs that respond to SOX2_124 and _216 are derived from four patients and are considered to be the most antigenic peptides.

Example  6

Cell injury analysis

(1) Injury to cells antigen-presenting SOX2-derived peptides

CD8 positive T cells were isolated from the peripheral blood lymphocytes of patient A in Example 5 in the same manner as in Example 2, and other mononuclear cells were added with phytohemagglutinin (PHA) to form agglutination. I was. The agglutinated monocytes were irradiated, and a mixture of 13 SOX2-derived peptides was added at a concentration of 10 µg / mL, 50 units of IL-2 were added, and cultured together with CD8 positive T cells. The same peptide stimulation was carried out three times per week, and 5 days after the third stimulation, as T2-A24 cells (T2-A24-SOX2) and negative control to which a mixture of T2-A24 cells and 13 SOX2 peptides was added. Cytotoxicity analysis was performed targeting human red sub-leukemia leukemia cells (K562).

Cytotoxicity measurement by cytotoxicity analysis was performed by ⁵¹ Cr release analysis. Target cells were labeled with 100 μCi of ⁵¹ Cr for 1 hour at 37 ° C. and washed three times in RPMI 1640 medium. Subsequently, target cells labeled with ⁵¹ Cr, together with effector cells, at various effector / target ratios (E / T ratios) at 37 ° C. for 6 hours, 96-well microtiter plates with V-type bottoms. Incubated at. Then, the supernatant was collected and radioactivity was measured with a gamma counter. Specific cell lysis (cytotoxic activity) was calculated in the same manner as in Example 3.

The results are shown in Figure 5 a). CD8 positive T cells mixed with SOX2 derived peptides specifically injured T2-A24 cells to which SOX2 derived peptides were added.

(2) Injury against SOX2 expressing cells

As in (1), HLA-A24 positive LHK2 lung cancer cells and SOX2 transgenic LHK2 cells (LHK2-SOX2) were targeted and cytotoxicity analysis was performed.

The result is shown in b) of FIG. CD8 positive T cells mixed with SOX2 derived peptides showed high cytotoxic activity against LHK2 cells forcibly expressing SOX2.

Cancer vaccines or anticancer agents based on the present invention can be an effective treatment or preventive means for cancers that were difficult to treat or prevent in conventional treatments. The present invention contributes to the improvement of medical care and human welfare.

                         SEQUENCE LISTING <110> Japan Science and Technology Agency       Sapporo Medical University <120> Molecular markers for cancer stem cells <130> PCT2393SI <160> 17 <170> PatentIn version 3.1 <210> 1 <211> 9 <212> PRT <213> Artificial <220> <223> pepitide fragment of SOX2 <400> 1 Met Tyr Asn Met Met Glu Thr Glu Leu 1 5 <210> 2 <211> 10 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 2 Val Trp Ser Arg Gly Gln Arg Arg Lys Met 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 3 Lys Met Ala Gln Glu Asn Pro Lys Met 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 4 Pro Phe Ile Asp Glu Ala Lys Arg Leu 1 5 <210> 5 <211> 11 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 5 Lys Tyr Arg Pro Arg Arg Lys Thr Lys Thr Leu 1 5 10 <210> 6 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 6 Leu Met Lys Lys Asp Lys Tyr Thr Leu 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 7 Lys Tyr Thr Leu Pro Gly Gly Leu Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 8 Gly Trp Ser Asn Gly Ser Tyr Ser Met 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 9 Ser Tyr Ser Met Met Gln Asp Gln Leu 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 10 Pro Met His Arg Tyr Asp Val Ser Ala Leu 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 11 Ser Met Thr Ser Ser Gln Thr Tyr Met 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 12 Tyr Met Asn Gly Ser Pro Thr Tyr Ser Met 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial <220> <223> peptide fragment of SOX2 <400> 13 Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial <220> <223> HLA-A24 binding epitope from HIV <400> 14 Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile 1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial <220> <223> HLA-A24 binding epitope from EBV <400> 15 Thr Tyr Gly Pro Val Phe Met Ser Leu 1 5 <210> 16 <211> 317 <212> PRT <213> Homo sapiens <400> 16 Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln 1 5 10 15 Thr Ser Gly Gly Gly Gly Gly Asn Ser Thr Ala Ala Ala Ala Gly Gly             20 25 30 Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn Ala Phe         35 40 45 Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu Asn Pro     50 55 60 Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys 65 70 75 80 Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala Lys Arg                 85 90 95 Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro             100 105 110 Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr Leu Pro         115 120 125 Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly Val Gly     130 135 140 Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp Ser Tyr 145 150 155 160 Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met Gln Asp                 165 170 175 Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly Ala Ala             180 185 190 Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn         195 200 205 Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser     210 215 220 Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly Ser Met 225 230 235 240 Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val Val Thr                 245 250 255 Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu Arg Asp             260 265 270 Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro Ala Ala         275 280 285 Pro Ser Arg Leu His Met Ser Gln His Tyr Gln Ser Gly Pro Val Pro     290 295 300 Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met 305 310 315 <210> 17 <211> 8 <212> PRT <213> Artificial <220> <223> Epitope from OVA <400> 17 Ser Ile Ile Asn Phe Glu Lys Leu 1 5

Claims (9)

A peptide capable of inducing cytotoxic T cells targeting cancer cells, wherein the peptide comprises an amino acid sequence derived from a polypeptide encoded by the Sox2 gene or one or several amino acids in the amino acid sequence. The peptide comprising an amino acid sequence deleted, substituted or added and antigenically presented by HLA-A24. The method of claim 1,
The peptide contains the amino acid sequence shown by sequence numbers 1, 4-9, and 12, or the sequence in which one or several amino acids were deleted, substituted, or added in the said amino acid sequence. A cytotoxic T cell induced by the peptide according to claim 1 or 2. DNA encoding the peptide according to claim 1. A pharmaceutical composition comprising the peptide of claim 1 or 2 and / or the cytotoxic T cell of claim 3 and / or the DNA of claim 4. 6. The method of claim 5,
A pharmaceutical composition wherein the pharmaceutical composition is a cancer vaccine. 6. The method of claim 5,
A pharmaceutical composition wherein the pharmaceutical composition is an anticancer agent. 6. The method of claim 5,
A pharmaceutical composition wherein the pharmaceutical composition is a DNA vaccine. Use of the peptide of Claim 1 or 2 for inducing cytotoxic T cells which target cancer cells.

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