WO2010107116A1 - Hla-a24-binding cancer antigen peptide derived from sox2 - Google Patents
Hla-a24-binding cancer antigen peptide derived from sox2 Download PDFInfo
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- WO2010107116A1 WO2010107116A1 PCT/JP2010/054852 JP2010054852W WO2010107116A1 WO 2010107116 A1 WO2010107116 A1 WO 2010107116A1 JP 2010054852 W JP2010054852 W JP 2010054852W WO 2010107116 A1 WO2010107116 A1 WO 2010107116A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Definitions
- the present invention relates to a peptide capable of inducing cytotoxic T cells (CTL) targeting cancer cells. Moreover, this invention relates to the cancer vaccine and anticancer agent containing the said peptide. The present invention further relates to the use of the peptide for inducing CTL targeting cancer cells, the resulting CTL and an anticancer agent comprising the CTL.
- CTL cytotoxic T cells
- cancer antigens have been identified in epithelial cancers such as colorectal cancer, stomach cancer, breast cancer, lung cancer, and bladder cancer that occupy the majority of cancers, and immunotherapy targeting cancer antigens has not been established. .
- the Sex determinig Region Y-box2 (Sox2) gene is expressed in the central nervous system of the embryonic period and is known as a gene related to the self-renewal of neural stem cells, but it is also highly expressed in malignant glioma I know.
- An announcement by M. Schmitz et al. Discloses that it is possible to artificially cause CTLs and eliminate gliomas using the HLA-A2-restricted epitope of Sox2 (Non-patent Document 8). ).
- Non-patent Document 8 Moreover, recent studies have shown that a large amount of Sox2 gene is expressed in cancer tissues including cancer stem cells that are considered to be the main cause of cancer recurrence and metastasis (Non-patent Document 1). ⁇ 7).
- An object of the present invention is to provide a cancer vaccine and an anticancer agent that can be used for cancer immunotherapy.
- CD8 (+) CTL recognizes and activates a complex composed of a major histocompatibility antigen complex (in humans, HLA) on cancer cells and a cancer antigen peptide presented on the HLA.
- HLA major histocompatibility antigen complex
- the activated CTL recognizes and attacks cancer cells via the T cell antigen receptor on the 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 CTLs to prevent and treat cancer.
- SOX2 is a gene transcription regulatory factor, and its 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 renal cancer, but expression in normal tissues is limited to fetal stem cells and neural stem cells.
- the present inventors have conducted extensive studies on cancer antigenicity, that is, CTL inducing ability of various SOX2-derived peptides. As a result, there is an HLA-A24 binding motif in the 317 amino acid sequence encoded by the SOX2 gene. We have found that certain peptides in this can induce CTL. These findings have led to the completion of the present invention.
- the present invention is a peptide capable of inducing cytotoxic T cells targeting cancer cells, wherein the peptide consists of an amino acid sequence derived from a polypeptide encoded by the Sox2 gene, or an amino acid sequence thereof In which one or several amino acids are deleted, substituted or added, and presented as an antigen by HLA-A24.
- the present invention also provides the peptide according to the above, wherein the peptide consists of the amino acid sequence shown in 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. Relates to peptides.
- the invention further relates to cytotoxic T cells induced by the above peptides.
- the present invention further relates to a DNA encoding the above peptide.
- the present invention also relates to a pharmaceutical composition comprising the above peptide and / or the above cytotoxic T cell and / or the above DNA. Furthermore, this invention relates to said pharmaceutical composition whose pharmaceutical composition is a cancer vaccine.
- the present invention also relates to the pharmaceutical composition as described above, wherein the pharmaceutical composition is an anticancer agent.
- the present invention further relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is a DNA vaccine.
- the present invention further relates to the use of the above peptides for inducing cytotoxic T cells targeting cancer cells.
- the modified peptide obtained by modifying the peptide of the present invention and 1-2 amino acids can induce CTLs that damage HLA-A24-positive SOX2-positive cancer cells that are thought to present the peptide of the present invention.
- the expression rate of HLA-A24 is 20-30% in Westerners and 50% or more in Japanese. Therefore, the cancer antigen peptide of the present invention can be used as a cancer vaccine and an anticancer agent useful for various types of SOX2-positive malignant tumors all over the world.
- FIG. 1 shows the results of an HLA-A24 binding assay for SOX2 polypeptide-derived peptides. Peptides that showed an MFI similar or close to that of the peptides used as positive controls were identified as HLA-A24 binding peptides.
- FIG. 2 shows the results of cytotoxicity assay of SOX2_109 peptide. Significantly large cytotoxicity has been detected against the negative control HIV-derived peptide.
- FIG. 3 shows the results of ELISPOT assay of SOX2_109 peptide. The HIV-derived peptide as a negative control did not detect the release of IFN ⁇ , whereas the SOX2 — 109 peptide confirmed the release of IFN ⁇ .
- FIG. 4 shows the results of ELISPOT assay of SOX2-derived peptides.
- sur2B represents a group added with survivin2B as a negative control, and ( ⁇ ) represents a group where no peptide was added.
- CTLs responding to SOX2_50 and _58 were not derived from any patient, and CTLs responding to SOX2_124 and _216 were derived from 4 patients.
- FIG. 5a) shows the results of cytotoxicity assay on T2-A24 cells presenting SOX2-derived peptides as antigens
- b) shows the results of cytotoxicity assays in LHK2 lung cancer cells and normal LHK2 cells forcibly expressing SOX.
- Identification of the peptides of the present invention involves the following steps: (1) providing a peptide derived from SOX2 polypeptide having a sequence corresponding to the binding motif of HLA-A24 which is human major histocompatibility complex (MHC) class I; (2) adding the peptide to an antigen-presenting cell expressing HLA-A24 to obtain an antigen-presenting cell presenting the peptide by HLA-A24; (3) stimulating T cells with the antigen-presenting cells to induce CTL, and (4) measuring the cancer cytotoxicity of the induced CTL, It can carry out by the method containing.
- MHC human major histocompatibility complex
- the peptide of the present invention Since the peptide of the present invention has as few as 10 amino acids, it can be synthesized by a general amino acid chemical synthesis method, for example, the Fmoc method. It can also be synthesized using a commercially available amino acid synthesizer.
- the peptide of the present invention is derived from SOX2 polypeptide expressed inside cancer cells, it can be obtained from cancer cells of cancer patients (Suzuki, K. et al., J. Immunol., 163, 2783-2791 ( 1999)), the cell surface HLA-binding peptide can be isolated to obtain the corresponding peptide.
- the peptides of the present invention include those obtained by optionally modifying one or more amino acids of the above peptides.
- the modified peptide can also cause CTL induction that damages cells that have presented the SOX2 polypeptide-derived peptide with HLA-A24.
- the peptide of the present invention can induce cytotoxic T cells targeting cancer cells.
- Cells expressing HLA-A24 used for induction may be collected from cancer patients, but may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.
- the peptide of the present invention may be any peptide derived from the SOX2 polypeptide, as long as it can induce CTLs that damage cells presented with antigen by HLA-A24, and has a certain binding affinity with HLA-A24.
- Peptides represented by SEQ ID NOs: 1, 4 to 9, and 11 are preferable in that they have sex.
- 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 DNA as long as it produces the peptide of the present invention when subjected to transcription and translation. Preferably, it encodes the peptide portion of the present invention in the Sox2 gene sequence. DNA having the same sequence as the target sequence.
- the peptide or DNA of the present invention can be used by itself or with an adjuvant, and further contains a pharmaceutically acceptable carrier as appropriate.
- Adjuvants include adjuvants intended to enhance the immune response, such as incomplete (complete) adjuvants of floyd, aluminum adjuvants, virus envelope vectors and the like.
- the pharmaceutically acceptable carrier include diluents such as PBS and distilled water, and physiological saline.
- the DNA used as the pharmaceutical composition includes a vector construct incorporating the DNA of the present invention in addition to the DNA alone.
- Any vector can be used as long as it allows the DNA of the present invention to act as a pharmaceutical composition, and a vector suitable for the form and purpose of the pharmaceutical composition can be appropriately employed by techniques well known in the art.
- the vector include, but are not limited to, a plasmid vector, a Sendai virus, 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.
- the cancer vaccine, anticancer agent or DNA vaccine of the present invention can be formed into a liquid, oil, emulsion, soft capsule, hard capsule, tablet, granule, solid agent or the like by a method well known in the art.
- the cancer vaccine and anticancer agent of the present invention can be administered orally, parenterally or transdermally depending on the form of use.
- intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration and the like can be mentioned.
- the dosage usually varies depending on the patient's body weight, the nature and condition of the disease, but is up to 5-10 mg per day for adult use.
- the dose is 0.1 to 10 mg per week, preferably 100 to 1000 ⁇ g.
- CTL obtained by using the peptide of the present invention targets cancer cells, it can be used as an anticancer agent.
- it may contain a pharmaceutically acceptable carrier as appropriate and can take various forms.
- the anticancer agent containing the CTL of the present invention can be administered parenterally in the same manner as the cancer vaccine and anticancer agent containing the peptide of the present invention.
- the cancer to which the cancer vaccine, anticancer agent and DNA vaccine of the present invention can be applied is a cancer comprising cancer cells presenting the peptide of the present invention with HLA-A24, for example, epithelial cancer. Examples of epithelial cancer include lung cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, breast cancer and the like.
- the peptides of the present invention can also be used to induce CTL targeting cancer cells.
- the induction can be performed according to the method described in the literature (Nabeta, Y. et al., Jpn. J. Cancer Res, 91, 616-621 (2000)). Specifically, the following steps: (1) providing a cell expressing HLA-A24; (2) A step of adding the peptide of the present invention to the cells and presenting the peptides on HLA-A24 (3) Stimulating T cells with cells presenting the peptides by HLA-A24, and treating the T cells with cancer Inducing cell target CTL, Can be used.
- Cells expressing HLA-A24 may be collected from cancer patients, but may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.
- Example 1 Sox2-derived peptide HLA-A24 peptide binding assay The amino acid sequence of Sox2 is shown in SEQ ID NO: 16. It is known that the peptide that binds to HLA-A24 has tyrosine, tryptophan, phenylalanine or methionine as the second amino acid, and leucine, isoleucine, tryptophan, phenylalanine or methionine as the C-terminal amino acid. Among sequences contained 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: PFIDAAKRL (SEQ ID NO: 4) SOX2 — 109: KYRPRRKTTKTL (SEQ ID NO: 5) SOX2 — 119: LMKKDKKYTL (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)
- T2-A24 cells are a cell line in which HLA-A2402 gene is introduced into human lymphoblastoid T2 cells and expressed.
- a low level of HLA-A24 molecule is expressed on the cell surface of this cell and can be detected using an HLA-A24-specific monoclonal antibody and a flow cytometer.
- the expression level is quantified as mean fluorescence intensity (MFI).
- MFI mean fluorescence intensity
- T2-A24 cells were cultured overnight at 26 ° C. Thereafter, the cells were washed with PBS, a synthetic peptide derived from SOX2, HIV peptide (SEQ ID NO: 14) derived from human immunodeficiency virus as a positive control, and EBV peptide (SEQ ID NO: 15) derived from Epstein-Barr virus.
- SL8 peptide SEQ ID NO: 17
- SL8 peptide which is a peptide derived from ovalbumin
- HLA-A24 antibody (C7709A2.6) was added to the isolated cells, and the mixture was allowed to stand at 4 ° C. for 1 hour, and then washed with PBS.
- a fluorescently labeled anti-mouse IgG + IgM antibody was added as a secondary antibody, and the mixture was allowed to stand at 4 ° C. for 30 minutes, and then 1% formalin was added to fix the cells.
- FITC fluorescence intensity was measured for the fixed cells with a flow cytometer (BD FACS Calibur).
- PBMC Peripheral blood mononuclear cells
- Treated cells were co-cultured with 20 U / ml IL-2 (Takeda Pharmaceutical Co., Ltd.), 100,000 cells / well of CD8 positive lymphocytes, stimulated with CD8 positive lymphocytes once a week, and stimulated 3 times in total. Induced.
- the peptide-specific reactivity of the induced CTL was evaluated by cytotoxicity assay or ELISPOT assay.
- Example 3 Cr 51 was added to the cytotoxicity assay T2A24, cultured for 1 hour at 37 ° C. in RPMI medium, radiolabeled, and then washed four times with RPMI medium.
- SOX2 peptide or control peptide HV peptide
- 2000 / well T2A24 pulsed with SOX2 peptide or control peptide and 14000 / well CD8 positive lymphocytes induced in Example 2 were co-cultured in RPMI medium at 37 ° C. for 4 hours. Thereafter, the supernatant was collected, and the gamma dose was measured with a gamma counter.
- the dose when cultivating peptide pulse T2A24 only with RPMI medium is spontaneously released (SPR), and the dose when cultivating cell lysate (2% NP40) and peptide pulse T2A24 is maximum released (MXR).
- SPR spontaneously released
- MXR maximum released
- the activity was calculated as (measured value ⁇ SPR) / (MXR ⁇ SPR) ⁇ 100.
- CTLs that specifically impaired T2A24 pulsed with SOX2 — 109 peptide were detected.
- the results are shown in FIG. In the HIV-derived peptide that is a control peptide, radiation derived from damaged cells was hardly detected, whereas in the SOX2_109 peptide, a large amount was detected.
- Example 4 ELISPOT assay experiments were performed using a Human IFN ⁇ ELISPOT set (BD).
- the ELISPOT plate was coated with the anti-IFN ⁇ antibody by standing overnight at 4 ° C.
- SOX2 peptide or control peptide (HIV peptide) was added to T2A24, and allowed to stand at room temperature for 1 hour.
- 50000 peptide-pulsed T2A24 and 10000 CTL derived in Example 2 were co-cultured overnight at 37 ° C. on a plate coated with anti-IFN ⁇ antibody. After washing with 1 ⁇ PBS and 0.05% Tween 20, a biotin-labeled anti-IFN ⁇ antibody was added and allowed to stand at room temperature for 2 hours.
- Example 5 Comparison of cytotoxic T cell induction ability of each SOX2-derived peptide Peripheral blood derived from 6 HLA-A2402-positive lung cancer patients was used. Informed consent is obtained from all patients. PBMCs were separated by conventional density gradient centrifugation with Lymphoprep® (Nycomed, Oslo, Norway).
- C1R lymphoma subcell line C1R-A24 cells expressing HLA-A24 were pulsed with 10 ⁇ g / mL of each of the SOX2 peptides and survivin2B peptide as a negative control. Thereafter, 5 ⁇ 10 3 CTLs were co-cultured with 5 ⁇ 10 4 antigen-presenting cells C1R-A24 cells. After 24 hours incubation at 37 ° C., 5% CO 2 , the wells were washed 5 times with PBS and incubated with biotinylated anti-human IFN- ⁇ antibody and horseradish peroxidase-conjugated avidin. The formed IFN ⁇ spots were counted using KS ELISPOT (Carl Zeiss).
- 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 Cytotoxicity assay (1) Toxicity to cells presenting antigens of SOX2-derived peptides CD8 positive T cells were isolated from peripheral blood lymphocytes of patient A in Example 5 in the same manner as in Example 2, and other mononuclear cells were separated. Added phytohemagglutinin (PHA) and allowed to blast. Irradiate blasted mononuclear cells, add a mixture of 13 SOX2-derived peptides at a concentration of 10 ⁇ g / mL, add 50 units of IL-2, and mix culture with CD8 positive T cells did.
- PHA phytohemagglutinin
- T2-A24 cells Similar peptide stimulation was performed 3 times per week, and 5 days after the third stimulation, T2-A24 cells, T2-A24 cells (T2-A24-SOX2) added with a mixture of 13 SOX2 peptides, and negative control A cytotoxic assay was conducted targeting human erythroblast-like leukemia cells (K562).
- Cytotoxicity measurement by cytotoxicity assays were performed by a 51 Cr release assay.
- Target cells were labeled with 100 ⁇ Ci 51 Cr for 1 hour at 37 ° C. and washed 3 times with RPMI 1640 medium.
- Target cells labeled with 51 Cr were then incubated with effector cells at various effector / target ratios (E / T ratio) at 37 ° C. for 6 hours in a V-bottom 96-well microtiter plate. Thereafter, the supernatant was collected and the radioactivity was measured with a gamma counter.
- % Specific cell lysis was calculated as in Example 3. The results are shown in FIG. CD8 positive T cells mixed and cultured with SOX2-derived peptides specifically damaged T2-A24 cells to which SOX2-derived peptides were added.
- the cancer vaccine or anticancer agent based on the present invention can be an effective treatment or prevention means for cancer that has been difficult to treat or prevent by conventional treatment methods.
- the present invention contributes to medical improvement and human welfare.
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Abstract
Disclosed are: a peptide capable of inducing a cytotoxic T cell that targets cancer cells, wherein the peptide comprises an amino acid sequence derived from a polypeptide encoded by Sox2 gene or an amino acid sequence produced by deleting, substituting or adding one or several amino acid residues in the aforementioned amino acid sequence and can be presented as an antigen in conjunction with HLA-A24; a CTL induced by the peptide; a pharmaceutical composition containing the peptide and/or the CTL; and use of the peptide for the induction of a CTL.
Description
本発明は、癌細胞を標的とする細胞傷害性Ｔ細胞（ＣＴＬ）を誘導することができるペプチドに関する。
また本発明は、前記ペプチドを含む癌ワクチン及び抗癌剤に関する。
更に本発明は、癌細胞を標的とするＣＴＬを誘導するための前記ペプチドの使用、得られたＣＴＬ及び前記ＣＴＬを含む抗癌剤に関する。 The present invention relates to a peptide capable of inducing cytotoxic T cells (CTL) targeting cancer cells.
Moreover, this invention relates to the cancer vaccine and anticancer agent containing the said peptide.
The present invention further relates to the use of the peptide for inducing CTL targeting cancer cells, the resulting CTL and an anticancer agent comprising the CTL.
また本発明は、前記ペプチドを含む癌ワクチン及び抗癌剤に関する。
更に本発明は、癌細胞を標的とするＣＴＬを誘導するための前記ペプチドの使用、得られたＣＴＬ及び前記ＣＴＬを含む抗癌剤に関する。 The present invention relates to a peptide capable of inducing cytotoxic T cells (CTL) targeting cancer cells.
Moreover, this invention relates to the cancer vaccine and anticancer agent containing the said peptide.
The present invention further relates to the use of the peptide for inducing CTL targeting cancer cells, the resulting CTL and an anticancer agent comprising the CTL.
近年における免疫学と分子生物学の進歩は、腫瘍免疫の進歩に多大の影響を与えている。ヒトでインフルエンザウイルス感染が起きた場合に、その感染に対して免疫が成立して感染症から離脱するという事象は以下の細胞性免疫により説明することができる。インフルエンザウイルスに感染した上皮細胞は、その細胞表面にある主要組織適合抗原複合体ＨＬＡ分子上にウイルスゲノム由来の９～１０のペプチドを提示する。このＨＬＡ－ウイルスペプチド複合体を提示する感染細胞は強烈なアロジェニック反応を惹起し、感染細胞は末梢血中に存在するＣＤ８陽性ＣＴＬにより特異的に認識され、積極的に排除される。この細胞性免疫のメカニズムは自己の細胞が腫瘍化して生じた癌細胞に対しても同様に働くと理解される。このことは、ベルギーのThierry Boonらによる悪性黒色腫からの腫瘍抗原ＭＡＧＥ遺伝子の単離により証明された（Van der Bruggen et al.、Science, 254, 1643-1647 (1991)）。
Recent advances in immunology and molecular biology have had a major impact on the advancement of tumor immunity. When influenza virus infection occurs in humans, the phenomenon of establishing immunity against the infection and withdrawing 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 complex HLA molecule on the cell surface. Infected cells presenting this HLA-viral peptide complex elicit a strong allogenic reaction, and the infected cells are specifically recognized by CD8 positive CTLs present in the peripheral blood and actively eliminated. It is understood that this mechanism of cell-mediated immunity works in the same manner against cancer cells that are generated by autologous cells becoming tumors. This was demonstrated by the isolation of the tumor antigen MAGE gene from malignant melanoma by Thierry Boon et al. In Belgium (Van der Bruggen et al., Science, 254 , 1643-1647 (1991)).
Ｔ細胞が認識する癌抗原の同定方法としては、Ｔ細胞を用いてヒト癌由来のｃＤＮＡライブラリーをスクリーニングする方法が既に開発されており、この方法を用いて前記のＭＡＧＥ遺伝子が単離された。それ以後、悪性黒色腫を初めとした癌細胞表面上のクラスＩ分子に提示されてＴ細胞に認識される癌由来癌抗原ペプチドが複数同定され、これらのいくつかを用いた臨床治験が開始されており、既に一定の成果が得られている。例えば、癌患者の血清中に存在する抗体により認識される分子として食道癌より同定されたＮＹ－ＥＳＯ－１分子は、その合成ペプチドがＣＴＬの誘導能を有することが認識されている（Chen, YT. et al.，Proc. Natl. Acad. USA, 94, 1914-1918 (1997)及びJager, E. et al.，J. Exp. Med., 187, 265-270(1998)）。
As a method for identifying cancer antigens recognized by T cells, a method for screening a cDNA library derived from human cancer using T cells has already been developed, and the above MAGE gene was isolated using this method. . Since then, several cancer-derived cancer antigen peptides, which are presented on class I molecules on the surface of cancer cells such as malignant melanoma and recognized by T cells, have been identified, and clinical trials using some of these have begun. And certain results have already been obtained. For example, the NY-ESO-1 molecule identified from esophageal cancer as a molecule recognized by an antibody present in the serum of a cancer patient is recognized to have a synthetic peptide capable of inducing 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)).
しかし、臨床的に癌の大部分を占める大腸癌、胃癌、乳癌、肺癌、膀胱癌等の上皮癌では癌抗原がほとんど同定されておらず、癌抗原を標的とした免疫療法は確立されていない。
However, few cancer antigens have been identified in epithelial cancers such as colorectal cancer, stomach cancer, breast cancer, lung cancer, and bladder cancer that occupy the majority of cancers, and immunotherapy targeting cancer antigens has not been established. .
Sex determinig Region Y-box2（Ｓｏｘ２）遺伝子は、胎生期の中枢神経に発現し、神経幹細胞の自己複製に関係する遺伝子として知られているが、悪性の神経膠腫においても多く発現していることがわかっている。M.Schmitzらの発表によって、Ｓｏｘ２のＨＬＡ－Ａ２拘束性エピトープを利用してＣＴＬ誘導を人為的に起こし、神経膠腫を排除することが可能であることが開示されている（非特許文献８）。
また、近年の研究で、癌の再発や転移の主要な原因と考えられている癌幹細胞を含む癌組織中においても、Ｓｏｘ２遺伝子が多く発現していることが示されている（非特許文献１～７）。 The Sex determinig Region Y-box2 (Sox2) gene is expressed in the central nervous system of the embryonic period and is known as a gene related to the self-renewal of neural stem cells, but it is also highly expressed in malignant glioma I know. An announcement by M. Schmitz et al. Discloses that it is possible to artificially cause CTLs and eliminate gliomas using the HLA-A2-restricted epitope of Sox2 (Non-patent Document 8). ).
Moreover, recent studies have shown that a large amount of Sox2 gene is expressed in cancer tissues including cancer stem cells that are considered to be the main cause of cancer recurrence and metastasis (Non-patent Document 1). ~ 7).
また、近年の研究で、癌の再発や転移の主要な原因と考えられている癌幹細胞を含む癌組織中においても、Ｓｏｘ２遺伝子が多く発現していることが示されている（非特許文献１～７）。 The Sex determinig Region Y-box2 (Sox2) gene is expressed in the central nervous system of the embryonic period and is known as a gene related to the self-renewal of neural stem cells, but it is also highly expressed in malignant glioma I know. An announcement by M. Schmitz et al. Discloses that it is possible to artificially cause CTLs and eliminate gliomas using the HLA-A2-restricted epitope of Sox2 (Non-patent Document 8). ).
Moreover, recent studies have shown that a large amount of Sox2 gene is expressed in cancer tissues including cancer stem cells that are considered to be the main cause of cancer recurrence and metastasis (Non-patent Document 1). ~ 7).
本発明は、癌の免疫療法に使用しうる癌ワクチン及び抗癌剤を提供することを目的とする。
An object of the present invention is to provide a cancer vaccine and an anticancer agent that can be used for cancer immunotherapy.
本発明者等は、免疫学的なヒト癌拒絶が主にＣＴＬ、特にＣＤ８(＋)ＣＴＬにより担われていることに着目した。ＣＤ８(＋)ＣＴＬは癌細胞上の主要組織適合抗原複合体（ヒトではＨＬＡ）と当該ＨＬＡ上に提示された癌抗原ペプチドとからなる複合体を認識して活性化する。そして、活性化されたＣＴＬはその細胞表面上のＴ細胞抗原レセプターを介して癌細胞を認識し、これを攻撃する。したがって、癌抗原ペプチドが同定されれば、これを癌ワクチン及び抗癌剤として使用し、ＣＴＬを効率的に誘導して、癌を予防及び治療することができる。
The inventors of the present invention focused on the fact that immunological human cancer rejection is mainly carried out by CTL, particularly CD8 (+) CTL. CD8 (+) CTL recognizes and activates a complex composed of a major histocompatibility antigen complex (in humans, HLA) on cancer cells and a cancer antigen peptide presented on the HLA. The activated CTL recognizes and attacks cancer cells via the T cell antigen receptor on the 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 CTLs to prevent and treat cancer.
ＳＯＸ２は遺伝子転写調節因子であり、癌細胞の中でも幹細胞様形質を有する癌幹細胞に特に発現レベルが高い。ＳＯＸ２は肺癌、腎癌等の多くの癌で発現するが、正常組織での発現は胎児性幹細胞と神経幹細胞等に限られている。
本発明者等は、種々のＳＯＸ２由来のペプチドについて癌抗原性、すなわちＣＴＬ誘導能について鋭意検討を重ねたところ、ＳＯＸ２遺伝子によりコードされる３１７個からなるアミノ酸配列内にＨＬＡ－Ａ２４結合モチーフがあり、このなかの特定のペプチドがＣＴＬを誘導することができることを見いだした。これらの知見により、本発明を完成するに至った。 SOX2 is a gene transcription regulatory factor, and its 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 renal cancer, but expression in normal tissues is limited to fetal stem cells and neural stem cells.
The present inventors have conducted extensive studies on cancer antigenicity, that is, CTL inducing ability of various SOX2-derived peptides. As a result, there is an HLA-A24 binding motif in the 317 amino acid sequence encoded by the SOX2 gene. We have found that certain peptides in this can induce CTL. These findings have led to the completion of the present invention.
本発明者等は、種々のＳＯＸ２由来のペプチドについて癌抗原性、すなわちＣＴＬ誘導能について鋭意検討を重ねたところ、ＳＯＸ２遺伝子によりコードされる３１７個からなるアミノ酸配列内にＨＬＡ－Ａ２４結合モチーフがあり、このなかの特定のペプチドがＣＴＬを誘導することができることを見いだした。これらの知見により、本発明を完成するに至った。 SOX2 is a gene transcription regulatory factor, and its 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 renal cancer, but expression in normal tissues is limited to fetal stem cells and neural stem cells.
The present inventors have conducted extensive studies on cancer antigenicity, that is, CTL inducing ability of various SOX2-derived peptides. As a result, there is an HLA-A24 binding motif in the 317 amino acid sequence encoded by the SOX2 gene. We have found that certain peptides in this can induce CTL. These findings have led to the completion of the present invention.
すなわち、本発明は、癌細胞を標的とする細胞傷害性Ｔ細胞を誘導しうるペプチドであって、該ペプチドが、Ｓｏｘ２遺伝子によってコードされるポリペプチド由来のアミノ酸配列からなるか、またはそのアミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されているアミノ酸配列からなり、かつＨＬＡ－Ａ２４によって抗原提示される、前記ペプチドに関する。
本発明はまた、ペプチドが、配列番号１、４～９および１２に示されるアミノ酸配列、または該アミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されている配列からなる、上記のペプチドに関する。
本発明はさらに、上記のペプチドにより誘導された細胞傷害性Ｔ細胞に関する。 That is, the present invention is a peptide capable of inducing cytotoxic T cells targeting cancer cells, wherein the peptide consists of an amino acid sequence derived from a polypeptide encoded by the Sox2 gene, or an amino acid sequence thereof In which one or several amino acids are deleted, substituted or added, and presented as an antigen by HLA-A24.
The present invention also provides the peptide according to the above, wherein the peptide consists of the amino acid sequence shown in 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. Relates to peptides.
The invention further relates to cytotoxic T cells induced by the above peptides.
本発明はまた、ペプチドが、配列番号１、４～９および１２に示されるアミノ酸配列、または該アミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されている配列からなる、上記のペプチドに関する。
本発明はさらに、上記のペプチドにより誘導された細胞傷害性Ｔ細胞に関する。 That is, the present invention is a peptide capable of inducing cytotoxic T cells targeting cancer cells, wherein the peptide consists of an amino acid sequence derived from a polypeptide encoded by the Sox2 gene, or an amino acid sequence thereof In which one or several amino acids are deleted, substituted or added, and presented as an antigen by HLA-A24.
The present invention also provides the peptide according to the above, wherein the peptide consists of the amino acid sequence shown in 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. Relates to peptides.
The invention further relates to cytotoxic T cells induced by the above peptides.
本発明はさらにまた、上記のペプチドをコードする、ＤＮＡに関する。
また、本発明は、上記のペプチドおよび／または上記の細胞傷害性Ｔ細胞および／または上記のＤＮＡを含む、医薬組成物に関する。
さらにまた、本発明は、医薬組成物が、癌ワクチンである、上記の医薬組成物に関する。
本発明はまた、医薬組成物が、抗癌剤である、上記の医薬組成物に関する。
本発明はさらに、医薬組成物が、ＤＮＡワクチンである、上記の医薬組成物に関する。
本発明はさらにまた、癌細胞を標的とする細胞傷害性Ｔ細胞を誘導するための、上記のペプチドの使用に関する。 The present invention further relates to a DNA encoding the above peptide.
The present invention also relates to a pharmaceutical composition comprising the above peptide and / or the above cytotoxic T cell and / or the above DNA.
Furthermore, this invention relates to said pharmaceutical composition whose pharmaceutical composition is a cancer vaccine.
The present invention also relates to the pharmaceutical composition as described above, wherein the pharmaceutical composition is an anticancer agent.
The present invention further relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is a DNA vaccine.
The present invention further relates to the use of the above peptides for inducing cytotoxic T cells targeting cancer cells.
また、本発明は、上記のペプチドおよび／または上記の細胞傷害性Ｔ細胞および／または上記のＤＮＡを含む、医薬組成物に関する。
さらにまた、本発明は、医薬組成物が、癌ワクチンである、上記の医薬組成物に関する。
本発明はまた、医薬組成物が、抗癌剤である、上記の医薬組成物に関する。
本発明はさらに、医薬組成物が、ＤＮＡワクチンである、上記の医薬組成物に関する。
本発明はさらにまた、癌細胞を標的とする細胞傷害性Ｔ細胞を誘導するための、上記のペプチドの使用に関する。 The present invention further relates to a DNA encoding the above peptide.
The present invention also relates to a pharmaceutical composition comprising the above peptide and / or the above cytotoxic T cell and / or the above DNA.
Furthermore, this invention relates to said pharmaceutical composition whose pharmaceutical composition is a cancer vaccine.
The present invention also relates to the pharmaceutical composition as described above, wherein the pharmaceutical composition is an anticancer agent.
The present invention further relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is a DNA vaccine.
The present invention further relates to the use of the above peptides for inducing cytotoxic T cells targeting cancer cells.
本発明のペプチドと１～２アミノ酸を改変した改変ペプチドは、本発明のペプチドを提示していると考えられるＨＬＡ－Ａ２４陽性ＳＯＸ２陽性の癌細胞を傷害するＣＴＬを誘導することができる。ＨＬＡ－Ａ２４の発現率は、欧米人では２０～３０％、日本人では５０％以上が陽性である。したがって、本発明の癌抗原ペプチドは世界中で、さまざまな種類のＳＯＸ２陽性の悪性腫瘍に対して有用な癌ワクチン及び抗癌剤として使用することができる。
The modified peptide obtained by modifying the peptide of the present invention and 1-2 amino acids can induce CTLs that damage HLA-A24-positive SOX2-positive cancer cells that are thought to present the peptide of the present invention. The expression rate of HLA-A24 is 20-30% in Westerners and 50% or more in Japanese. Therefore, the cancer antigen peptide of the present invention can be used as a cancer vaccine and an anticancer agent useful for various types of SOX2-positive malignant tumors all over the world.
以下、本発明について詳細に説明する。
本発明のペプチドの同定は、以下の工程：
（１）ヒト主要組織適合抗原複合体（ＭＨＣ）クラスIであるＨＬＡ－Ａ２４の結合モチーフに対応する配列を有する、ＳＯＸ２ポリペプチド由来のペプチドを提供する工程、
（２）前記のペプチドを、ＨＬＡ－Ａ２４を発現する抗原提示細胞に添加しＨＬＡ－Ａ２４により前記ペプチドを提示している抗原提示細胞を得る工程、
（３）前記抗原提示細胞でＴ細胞を刺激してＣＴＬを誘導する工程、及び、
（４）誘導されたＣＴＬの癌細胞傷害能を測定する工程、
を含む方法により行うことができる。 Hereinafter, the present invention will be described in detail.
Identification of the peptides of the present invention involves the following steps:
(1) providing a peptide derived from SOX2 polypeptide having a sequence corresponding to the binding motif of HLA-A24 which is human major histocompatibility complex (MHC) class I;
(2) adding the peptide to an antigen-presenting cell expressing HLA-A24 to obtain an antigen-presenting cell presenting the peptide by HLA-A24;
(3) stimulating T cells with the antigen-presenting cells to induce CTL, and
(4) measuring the cancer cytotoxicity of the induced CTL,
It can carry out by the method containing.
本発明のペプチドの同定は、以下の工程：
（１）ヒト主要組織適合抗原複合体（ＭＨＣ）クラスIであるＨＬＡ－Ａ２４の結合モチーフに対応する配列を有する、ＳＯＸ２ポリペプチド由来のペプチドを提供する工程、
（２）前記のペプチドを、ＨＬＡ－Ａ２４を発現する抗原提示細胞に添加しＨＬＡ－Ａ２４により前記ペプチドを提示している抗原提示細胞を得る工程、
（３）前記抗原提示細胞でＴ細胞を刺激してＣＴＬを誘導する工程、及び、
（４）誘導されたＣＴＬの癌細胞傷害能を測定する工程、
を含む方法により行うことができる。 Hereinafter, the present invention will be described in detail.
Identification of the peptides of the present invention involves the following steps:
(1) providing a peptide derived from SOX2 polypeptide having a sequence corresponding to the binding motif of HLA-A24 which is human major histocompatibility complex (MHC) class I;
(2) adding the peptide to an antigen-presenting cell expressing HLA-A24 to obtain an antigen-presenting cell presenting the peptide by HLA-A24;
(3) stimulating T cells with the antigen-presenting cells to induce CTL, and
(4) measuring the cancer cytotoxicity of the induced CTL,
It can carry out by the method containing.
本発明のペプチドはアミノ酸数が１０と小さいので、一般的なアミノ酸の化学合成法、例えばＦｍｏｃ法により合成することができる。市販のアミノ酸合成装置を使用して合成することもできる。また、本発明のペプチドは、癌細胞の内部に発現しているＳＯＸ２ポリペプチドに由来するので、癌患者の癌細胞から文献（Suzuki, K.ら，J. Immunol., 163, 2783-2791 (1999)）に記載の方法にしたがい細胞表面ＨＬＡ結合ペプチドを単離して、該当するペプチドを得ることもできる。
Since the peptide of the present invention has as few as 10 amino acids, it can be synthesized by a general amino acid chemical synthesis method, for example, the Fmoc method. It can also be synthesized using a commercially available amino acid synthesizer. In addition, since the peptide of the present invention is derived from SOX2 polypeptide expressed inside cancer cells, it can be obtained from cancer cells of cancer patients (Suzuki, K. et al., J. Immunol., 163, 2783-2791 ( 1999)), the cell surface HLA-binding peptide can be isolated to obtain the corresponding peptide.
本発明のペプチドには、上記のペプチドの１または複数のアミノ酸を随意に改変したものが含まれる。改変ペプチドもまた、ＨＬＡ－Ａ２４によってＳＯＸ２ポリペプチド由来ペプチドを抗原提示した細胞を傷害するＣＴＬ誘導を起こすことが可能である。
The peptides of the present invention include those obtained by optionally modifying one or more amino acids of the above peptides. The modified peptide can also cause CTL induction that damages cells that have presented the SOX2 polypeptide-derived peptide with HLA-A24.
本発明のペプチドによって、癌細胞を標的とする細胞傷害性Ｔ細胞を誘導することができる。誘導するために使用するＨＬＡ－Ａ２４を発現する細胞は、癌患者から採取したものでもよいが、非ＨＬＡ－Ａ２４発現細胞に、ＨＬＡ－Ａ２４をコードする遺伝子を導入して作成してもよい。
The peptide of the present invention can induce cytotoxic T cells targeting cancer cells. Cells expressing HLA-A24 used for induction may be collected from cancer patients, but may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.
本発明のペプチドとしては、ＳＯＸ２ポリペプチド由来のペプチドであって、ＨＬＡ－Ａ２４によって抗原提示した細胞を傷害するＣＴＬを誘導することができるものであれば何でもよく、ＨＬＡ－Ａ２４とある程度の結合親和性を有するという点で、好ましくは配列番号１、４～９および１１で表されるペプチドである。
The peptide of the present invention may be any peptide derived from the SOX2 polypeptide, as long as it can induce CTLs that damage cells presented with antigen by HLA-A24, and has a certain binding affinity with HLA-A24. Peptides represented by SEQ ID NOs: 1, 4 to 9, and 11 are preferable in that they have sex.
本発明のペプチドをコードするＤＮＡも、本発明に含まれる。本発明のペプチドをコードするＤＮＡは、転写、翻訳を受けた際に、本発明のペプチドを産生するものであれば何でもよいが、好ましくは、Ｓｏｘ２遺伝子配列のうち、本発明のペプチド部分をコードする配列と同じ配列を有するＤＮＡである。
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 DNA as long as it produces the peptide of the present invention when subjected to transcription and translation. Preferably, it encodes the peptide portion of the present invention in the Sox2 gene sequence. DNA having the same sequence as the target sequence.
本発明のペプチドまたはＤＮＡを医薬組成物として使用する場合、本発明のペプチドまたはＤＮＡは、それ自身で又は補助剤と共に使用することができ、更に医薬的に許容しうる担体を適宜含有させることができる。補助剤としては、免疫応答の強化を目的とするアジュバント、例えばフロイドの不完全（完全）アジュバント、アルミニウムアジュバント、ウィルスエンベロープベクター等が挙げられる。
医薬的に許容しうる担体としては、例えばＰＢＳ、蒸留水等の希釈剤、生理食塩水等が挙げられる。 When the peptide or DNA of the present invention is used as a pharmaceutical composition, the peptide or DNA of the present invention can be used by itself or with an adjuvant, and further contains a pharmaceutically acceptable carrier as appropriate. it can. Adjuvants include adjuvants intended to enhance the immune response, such as incomplete (complete) adjuvants of floyd, aluminum adjuvants, virus envelope vectors and the like.
Examples of the pharmaceutically acceptable carrier include diluents such as PBS and distilled water, and physiological saline.
医薬的に許容しうる担体としては、例えばＰＢＳ、蒸留水等の希釈剤、生理食塩水等が挙げられる。 When the peptide or DNA of the present invention is used as a pharmaceutical composition, the peptide or DNA of the present invention can be used by itself or with an adjuvant, and further contains a pharmaceutically acceptable carrier as appropriate. it can. Adjuvants include adjuvants intended to enhance the immune response, such as incomplete (complete) adjuvants of floyd, aluminum adjuvants, virus envelope vectors and the like.
Examples of the pharmaceutically acceptable carrier include diluents such as PBS and distilled water, and physiological saline.
医薬組成物として使用されるＤＮＡには、ＤＮＡ単体のほかに、本発明のＤＮＡを組み込んだベクターコンストラクトも含まれる。ベクターとしては、本発明のＤＮＡを医薬組成物として作用させることができるものであれば何でもよく、当該技術分野において周知の技術によって、医薬組成物の形態や目的に適したベクターが適宜採用され得る。ベクターとしては、これに限定されるものではないが、例えばプラスミドベクター、センダイウィルス、レトロウィルス、ワクシニアウィルス、アデノウィルス、アデノ随伴ウィルス、ヘルペスウィルス、インフルエンザウィルスなどに由来するウィルスベクターなどが挙げられる。
The DNA used as the pharmaceutical composition includes a vector construct incorporating the DNA of the present invention in addition to the DNA alone. Any vector can be used as long as it allows the DNA of the present invention to act as a pharmaceutical composition, and a vector suitable for the form and purpose of the pharmaceutical composition can be appropriately employed by techniques well known in the art. . Examples of the vector include, but are not limited to, a plasmid vector, a Sendai virus, a retrovirus, a vaccinia virus, an adenovirus, an adeno-associated virus, a herpes virus, an influenza virus, and the like.
本発明の医薬組成物は、癌ワクチン、抗癌剤またはＤＮＡワクチンなどの形態で使用することができる。本発明の癌ワクチン、抗癌剤またはＤＮＡワクチンは、当該技術分野において周知の方法により、液剤、油剤、エマルジョン、ソフトカプセル剤、ハードカプセル剤、錠剤、顆粒剤、固形剤等の形態にすることができる。
本発明の癌ワクチン及び抗癌剤は、その使用形態に応じて経口、非経口又は経皮投与することができる。例えば、静注投与、筋注投与、皮下投与、皮内投与等が挙げられる。投与量は、通常、患者の体重、疾患の性質及び状態に依存して変化するが、成人に使用する場合、１日あたり最大で５～１０ｍｇである。例えば、成人癌患者に皮下注射により使用する場合、1週間あたり０．１～１０ｍｇであり、好ましくは１００～１０００μｇである。 The pharmaceutical composition of the present invention can be used in the form of a cancer vaccine, an anticancer agent or a DNA vaccine. The cancer vaccine, anticancer agent or DNA vaccine of the present invention can be formed into a liquid, oil, emulsion, soft capsule, hard capsule, tablet, granule, solid agent or the like by a method well known in the art.
The cancer vaccine and anticancer agent of the present invention can be administered orally, parenterally or transdermally depending on the form of use. For example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration and the like can be mentioned. The dosage usually varies depending on the patient's body weight, the nature and condition of the disease, but is up to 5-10 mg per day for adult use. For example, when used by subcutaneous injection in adult cancer patients, the dose is 0.1 to 10 mg per week, preferably 100 to 1000 μg.
本発明の癌ワクチン及び抗癌剤は、その使用形態に応じて経口、非経口又は経皮投与することができる。例えば、静注投与、筋注投与、皮下投与、皮内投与等が挙げられる。投与量は、通常、患者の体重、疾患の性質及び状態に依存して変化するが、成人に使用する場合、１日あたり最大で５～１０ｍｇである。例えば、成人癌患者に皮下注射により使用する場合、1週間あたり０．１～１０ｍｇであり、好ましくは１００～１０００μｇである。 The pharmaceutical composition of the present invention can be used in the form of a cancer vaccine, an anticancer agent or a DNA vaccine. The cancer vaccine, anticancer agent or DNA vaccine of the present invention can be formed into a liquid, oil, emulsion, soft capsule, hard capsule, tablet, granule, solid agent or the like by a method well known in the art.
The cancer vaccine and anticancer agent of the present invention can be administered orally, parenterally or transdermally depending on the form of use. For example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration and the like can be mentioned. The dosage usually varies depending on the patient's body weight, the nature and condition of the disease, but is up to 5-10 mg per day for adult use. For example, when used by subcutaneous injection in adult cancer patients, the dose is 0.1 to 10 mg per week, preferably 100 to 1000 μg.
また、本発明のペプチドを使用して得られたＣＴＬは癌細胞を標的とするので、これを抗癌剤として使用することができる。この場合、前記の本発明のペプチドを含む抗癌剤と同様に、適宜医薬的に許容しうる担体を含み、かつ種々の形態をとることができる。本発明のＣＴＬを含む抗癌剤は、本発明のペプチドを含む癌ワクチン及び抗癌剤と同様に非経口投与することができる。
本発明の癌ワクチン、抗癌剤およびＤＮＡワクチンを適用しうる癌は、本発明のペプチドをＨＬＡ－Ａ２４により提示している癌細胞からなる癌、例えば上皮癌である。上皮癌としては、肺癌、胃癌、大腸癌、膀胱癌、膵癌、前立腺癌、乳癌等が挙げられる。 Moreover, since CTL obtained by 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, it may contain a pharmaceutically acceptable carrier as appropriate and can take various forms. The anticancer agent containing the CTL of the present invention can be administered parenterally in the same manner as the cancer vaccine and anticancer agent containing the peptide of the present invention.
The cancer to which the cancer vaccine, anticancer agent and DNA vaccine of the present invention can be applied is a cancer comprising cancer cells presenting the peptide of the present invention with HLA-A24, for example, epithelial cancer. Examples of epithelial cancer include lung cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, breast cancer and the like.
本発明の癌ワクチン、抗癌剤およびＤＮＡワクチンを適用しうる癌は、本発明のペプチドをＨＬＡ－Ａ２４により提示している癌細胞からなる癌、例えば上皮癌である。上皮癌としては、肺癌、胃癌、大腸癌、膀胱癌、膵癌、前立腺癌、乳癌等が挙げられる。 Moreover, since CTL obtained by 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, it may contain a pharmaceutically acceptable carrier as appropriate and can take various forms. The anticancer agent containing the CTL of the present invention can be administered parenterally in the same manner as the cancer vaccine and anticancer agent containing the peptide of the present invention.
The cancer to which the cancer vaccine, anticancer agent and DNA vaccine of the present invention can be applied is a cancer comprising cancer cells presenting the peptide of the present invention with HLA-A24, for example, epithelial cancer. Examples of epithelial cancer include lung cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, breast cancer and the like.
また、本発明のペプチドを、癌細胞を標的とするＣＴＬを誘導するために使用することができる。誘導は、例えば文献（Nabeta, Y.ら，Jpn. J. Cancer Res, 91, 616-621 (2000)）に記載の方法にしたがい行うことができる。
具体的には以下の工程：
（１）ＨＬＡ－Ａ２４を発現している細胞を提供する工程、
（２）前記細胞に本発明のペプチドを添加して、ＨＬＡ－Ａ２４上に提示させる工程
（３）前記ペプチドをＨＬＡ－Ａ２４により提示している細胞でＴ細胞を刺激し、前記Ｔ細胞を癌細胞標的ＣＴＬへ誘導する工程、
を含む方法を使用することができる。
ＨＬＡ－Ａ２４を発現する細胞は癌患者から採取したものでもよいが、非ＨＬＡ－Ａ２４発現細胞に、ＨＬＡ－Ａ２４をコードする遺伝子を導入して作成してもよい。 The peptides of the present invention can also be used to induce CTL targeting cancer cells. The induction can be performed according to the method described in the literature (Nabeta, Y. et al., Jpn. J. Cancer Res, 91, 616-621 (2000)).
Specifically, the following steps:
(1) providing a cell expressing HLA-A24;
(2) A step of adding the peptide of the present invention to the cells and presenting the peptides on HLA-A24 (3) Stimulating T cells with cells presenting the peptides by HLA-A24, and treating the T cells with cancer Inducing cell target CTL,
Can be used.
Cells expressing HLA-A24 may be collected from cancer patients, but may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.
具体的には以下の工程：
（１）ＨＬＡ－Ａ２４を発現している細胞を提供する工程、
（２）前記細胞に本発明のペプチドを添加して、ＨＬＡ－Ａ２４上に提示させる工程
（３）前記ペプチドをＨＬＡ－Ａ２４により提示している細胞でＴ細胞を刺激し、前記Ｔ細胞を癌細胞標的ＣＴＬへ誘導する工程、
を含む方法を使用することができる。
ＨＬＡ－Ａ２４を発現する細胞は癌患者から採取したものでもよいが、非ＨＬＡ－Ａ２４発現細胞に、ＨＬＡ－Ａ２４をコードする遺伝子を導入して作成してもよい。 The peptides of the present invention can also be used to induce CTL targeting cancer cells. The induction can be performed according to the method described in the literature (Nabeta, Y. et al., Jpn. J. Cancer Res, 91, 616-621 (2000)).
Specifically, the following steps:
(1) providing a cell expressing HLA-A24;
(2) A step of adding the peptide of the present invention to the cells and presenting the peptides on HLA-A24 (3) Stimulating T cells with cells presenting the peptides by HLA-A24, and treating the T cells with cancer Inducing cell target CTL,
Can be used.
Cells expressing HLA-A24 may be collected from cancer patients, but may be prepared by introducing a gene encoding HLA-A24 into non-HLA-A24 expressing cells.
以下の実施例は本発明について、さらに具体的に説明するものであり、本発明の範囲を何ら限定するものではない。当業者として通常の知識および技術を有するものは、本発明の精神を逸脱しない範囲で、下記実施例で示された態様に多様な改変を行うことができるが、かかる改変された態様も本発明に含まれる。
The following examples are intended to explain the present invention more specifically and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications to the embodiments shown in the following examples without departing from the spirit of the present invention without departing from the spirit of the present invention. include.
実施例１
Ｓｏｘ２由来ペプチドのＨＬＡ－Ａ２４ペプチド結合アッセイ
Ｓｏｘ２のアミノ酸配列は、配列番号１６に示されている。ＨＬＡ－Ａ２４に結合するペプチドは、２番目のアミノ酸がチロシン、トリプトファン、フェニルアラニンまたはメチオニンであり、Ｃ末端のアミノ酸がロイシン、イソロイシン、トリプトファン、フェニルアラニンまたはメチオニンであることが知られている。Ｓｏｘ２のアミノ酸配列に含まれる配列のうち、このＨＬＡ－Ａ２４結合モチーフを有する９から１１アミノ酸配列を選択し、以下のペプチドを計１３種類合成した。 Example 1
Sox2-derived peptide HLA-A24 peptide binding assay The amino acid sequence of Sox2 is shown in SEQ ID NO: 16. It is known that the peptide that binds to HLA-A24 has tyrosine, tryptophan, phenylalanine or methionine as the second amino acid, and leucine, isoleucine, tryptophan, phenylalanine or methionine as the C-terminal amino acid. Among sequences contained 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由来ペプチドのＨＬＡ－Ａ２４ペプチド結合アッセイ
Ｓｏｘ２のアミノ酸配列は、配列番号１６に示されている。ＨＬＡ－Ａ２４に結合するペプチドは、２番目のアミノ酸がチロシン、トリプトファン、フェニルアラニンまたはメチオニンであり、Ｃ末端のアミノ酸がロイシン、イソロイシン、トリプトファン、フェニルアラニンまたはメチオニンであることが知られている。Ｓｏｘ２のアミノ酸配列に含まれる配列のうち、このＨＬＡ－Ａ２４結合モチーフを有する９から１１アミノ酸配列を選択し、以下のペプチドを計１３種類合成した。 Example 1
Sox2-derived peptide HLA-A24 peptide binding assay The amino acid sequence of Sox2 is shown in SEQ ID NO: 16. It is known that the peptide that binds to HLA-A24 has tyrosine, tryptophan, phenylalanine or methionine as the second amino acid, and leucine, isoleucine, tryptophan, phenylalanine or methionine as the C-terminal amino acid. Among sequences contained 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（配列番号１）
ＳＯＸ２＿５０：ＶＷＳＲＧＱＲＲＫＭ（配列番号２）
ＳＯＸ２＿５８：ＫＭＡＱＥＮＰＫＭ（配列番号３）
ＳＯＸ２＿８９：ＰＦＩＤＥＡＫＲＬ（配列番号４）
ＳＯＸ２＿１０９：ＫＹＲＰＲＲＫＴＫＴＬ（配列番号５）
ＳＯＸ２＿１１９：ＬＭＫＫＤＫＹＴＬ（配列番号６）
ＳＯＸ２＿１２４：ＫＹＴＬＰＧＧＬＬ（配列番号７）
ＳＯＸ２＿１６５：ＧＷＳＮＧＳＹＳＭ（配列番号８）
ＳＯＸ２＿１７０：ＳＹＳＭＭＱＤＱＬ（配列番号９）
ＳＯＸ２＿１９６：ＰＭＨＲＹＤＶＳＡＬ（配列番号１０）
ＳＯＸ２＿２０９：ＳＭＴＳＳＱＴＹＭ（配列番号１１）
ＳＯＸ２＿２１６：ＹＭＮＧＳＰＴＹＳＭ（配列番号１２）
ＳＯＸ２＿２２６：ＳＹＳＱＱＧＴＰＧＭ（配列番号１３） SOX2_1: MYNMMETEL (SEQ ID NO: 1)
SOX2_50: VWSRGQRRKM (SEQ ID NO: 2)
SOX2_58: KMAQENPKM (SEQ ID NO: 3)
SOX2 — 89: PFIDAAKRL (SEQ ID NO: 4)
SOX2 — 109: KYRPRRKTTKTL (SEQ ID NO: 5)
SOX2 — 119: LMKKDKKYTL (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)
ＳＯＸ２＿５０：ＶＷＳＲＧＱＲＲＫＭ（配列番号２）
ＳＯＸ２＿５８：ＫＭＡＱＥＮＰＫＭ（配列番号３）
ＳＯＸ２＿８９：ＰＦＩＤＥＡＫＲＬ（配列番号４）
ＳＯＸ２＿１０９：ＫＹＲＰＲＲＫＴＫＴＬ（配列番号５）
ＳＯＸ２＿１１９：ＬＭＫＫＤＫＹＴＬ（配列番号６）
ＳＯＸ２＿１２４：ＫＹＴＬＰＧＧＬＬ（配列番号７）
ＳＯＸ２＿１６５：ＧＷＳＮＧＳＹＳＭ（配列番号８）
ＳＯＸ２＿１７０：ＳＹＳＭＭＱＤＱＬ（配列番号９）
ＳＯＸ２＿１９６：ＰＭＨＲＹＤＶＳＡＬ（配列番号１０）
ＳＯＸ２＿２０９：ＳＭＴＳＳＱＴＹＭ（配列番号１１）
ＳＯＸ２＿２１６：ＹＭＮＧＳＰＴＹＳＭ（配列番号１２）
ＳＯＸ２＿２２６：ＳＹＳＱＱＧＴＰＧＭ（配列番号１３） SOX2_1: MYNMMETEL (SEQ ID NO: 1)
SOX2_50: VWSRGQRRKM (SEQ ID NO: 2)
SOX2_58: KMAQENPKM (SEQ ID NO: 3)
SOX2 — 89: PFIDAAKRL (SEQ ID NO: 4)
SOX2 — 109: KYRPRRKTTKTL (SEQ ID NO: 5)
SOX2 — 119: LMKKDKKYTL (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細胞はヒトリンパ芽球様細胞Ｔ２細胞にＨＬＡ－Ａ２４０２遺伝子を導入して発現させた細胞株である。この細胞の細胞表面には低レベルのＨＬＡ－Ａ２４分子が発現しており、ＨＬＡ－Ａ２４特異的モノクローナル抗体とフローサイトメーターを用いて検出することができる。発現レベルは平均蛍光強度(ＭＦＩ)として定量化される。この細胞に試験管内で合成ペプチドを添加すると、添加したペプチドがＨＬＡ－Ａ２４分子と結合する場合、結合親和性と相関して細胞表面ＨＬＡ－Ａ２４発現レベルが増加する。この実験系を用いて、本発明のＳＯＸ２由来癌抗原ペプチドのＨＬＡ－Ａ２４結合親和性を解析した。
T2-A24 cells are a cell line in which HLA-A2402 gene is introduced into human lymphoblastoid T2 cells and expressed. A low level of HLA-A24 molecule is expressed on the cell surface of this cell and can be detected using an HLA-A24-specific monoclonal antibody and a flow cytometer. The expression level is quantified as mean fluorescence intensity (MFI). When a synthetic peptide is added to the cells in vitro, when the added peptide binds to the HLA-A24 molecule, the cell surface HLA-A24 expression level increases in correlation with the binding affinity. Using this experimental system, the HLA-A24 binding affinity of the SOX2-derived cancer antigen peptide of the present invention was analyzed.
Ｔ２－Ａ２４細胞を、２６℃で一晩培養した。その後細胞をＰＢＳで洗浄し、ＳＯＸ２由来の合成ペプチド、ポジティブコントロールとしてヒト免疫不全ウィルス由来のペプチドであるＨＩＶペプチド（配列番号１４）、エプスタイン－バーウィルス由来のペプチドであるＥＢＶペプチド（配列番号１５）、およびネガティブコントロールとして卵白アルブミン由来のペプチドであるＳＬ８ペプチド（配列番号１７）を加えて、２６℃で３時間共培養した。温度を３７℃にしてさらに２．５時間共培養した後、遠心分離して上清を除き、細胞を単離した。単離した細胞にＨＬＡ－Ａ２４抗体（Ｃ７７０９Ａ２．６）を加え、４℃で１時間静置し、その後ＰＢＳで洗浄した。２次抗体として蛍光標識抗マウスＩｇＧ＋ＩｇＭ抗体を加え、４℃で３０分静置した後、１％ホルマリンを加えて細胞を固定した。固定した細胞を、フローサイトメーター（BD FACS Calibur）にて、ＦＩＴＣ蛍光強度を計測した。
T2-A24 cells were cultured overnight at 26 ° C. Thereafter, the cells were washed with PBS, a synthetic peptide derived from SOX2, HIV peptide (SEQ ID NO: 14) derived from human immunodeficiency virus as a positive control, and EBV peptide (SEQ ID NO: 15) derived from Epstein-Barr virus. As a negative control, SL8 peptide (SEQ ID NO: 17), which is a peptide derived from ovalbumin, was added and co-cultured at 26 ° C. for 3 hours. After further incubation for 2.5 hours at a temperature of 37 ° C., the supernatant was removed by centrifugation, and the cells were isolated. HLA-A24 antibody (C7709A2.6) was added to the isolated cells, and the mixture was allowed to stand at 4 ° C. for 1 hour, and then washed with PBS. A fluorescently labeled anti-mouse IgG + IgM antibody was added as a secondary antibody, and the mixture was allowed to stand at 4 ° C. for 30 minutes, and then 1% formalin was added to fix the cells. FITC fluorescence intensity was measured for the fixed cells with a flow cytometer (BD FACS Calibur).
結果は図１に示されている。ポジティブコントロールと同等以上か、それに近い蛍光強度を示した８個のＳＯＸ２ペプチドを、ＨＬＡ－Ａ２４結合性ペプチドと判定した。
The result is shown in FIG. Eight SOX2 peptides showing fluorescence intensities that were equal to or higher than those of the positive control were determined as HLA-A24 binding peptides.
実施例２
ＣＴＬ誘導
インフォームドコンセントを得たＨＬＡ－Ａ２４陽性肺がん患者あるいはＨＬＡ－Ａ２４陽性健常者の末梢血５０ｍｌから、フィコール・コンレイ密度勾配中で遠心することにより、末梢血単核球（ＰＢＭＣ）を分離、回収した。続いてＣＤ８ ＭＡＣＳ ｂｅａｄｓを用いて、ＰＢＭＣよりＣＤ８陽性リンパ球とＣＤ８陰性リンパ球に分離した。
９６ウェルプレートで２０００００個／ウェルのＣＤ８陰性リンパ球と上記ＨＬＡ－Ａ２４結合性ＳＯＸ２ペプチドをそれぞれ混ぜ、室温で２時間静置後、１００Ｇｙ分放射線処理した。処理した細胞を２０Ｕ／ｍｌのＩＬ－２（武田薬品工業）、１０００００個／ウェルのＣＤ８陽性リンパ球と共培養し、ＣＤ８陽性リンパ球刺激を週１回行い、計３回刺激してＣＴＬを誘導した。誘導したＣＴＬのペプチド特異的反応性を細胞傷害アッセイあるいはＥＬＩＳＰＯＴアッセイにて評価した。 Example 2
Peripheral blood mononuclear cells (PBMC) were separated from 50 ml of peripheral blood of HLA-A24-positive lung cancer patients or HLA-A24-positive healthy subjects who obtained CTL-derived informed consent by centrifugation in a Ficoll-Conlay density gradient. It was collected. Subsequently, CD8 MACS beads were used to separate CD8 positive lymphocytes and CD8 negative lymphocytes from PBMC.
In a 96-well plate, 200000 CD8 negative lymphocytes and the above HLA-A24-binding SOX2 peptide were mixed, allowed to stand at room temperature for 2 hours, and then subjected to radiation treatment for 100 Gy. Treated cells were co-cultured with 20 U / ml IL-2 (Takeda Pharmaceutical Co., Ltd.), 100,000 cells / well of CD8 positive lymphocytes, stimulated with CD8 positive lymphocytes once a week, and stimulated 3 times in total. Induced. The peptide-specific reactivity of the induced CTL was evaluated by cytotoxicity assay or ELISPOT assay.
ＣＴＬ誘導
インフォームドコンセントを得たＨＬＡ－Ａ２４陽性肺がん患者あるいはＨＬＡ－Ａ２４陽性健常者の末梢血５０ｍｌから、フィコール・コンレイ密度勾配中で遠心することにより、末梢血単核球（ＰＢＭＣ）を分離、回収した。続いてＣＤ８ ＭＡＣＳ ｂｅａｄｓを用いて、ＰＢＭＣよりＣＤ８陽性リンパ球とＣＤ８陰性リンパ球に分離した。
９６ウェルプレートで２０００００個／ウェルのＣＤ８陰性リンパ球と上記ＨＬＡ－Ａ２４結合性ＳＯＸ２ペプチドをそれぞれ混ぜ、室温で２時間静置後、１００Ｇｙ分放射線処理した。処理した細胞を２０Ｕ／ｍｌのＩＬ－２（武田薬品工業）、１０００００個／ウェルのＣＤ８陽性リンパ球と共培養し、ＣＤ８陽性リンパ球刺激を週１回行い、計３回刺激してＣＴＬを誘導した。誘導したＣＴＬのペプチド特異的反応性を細胞傷害アッセイあるいはＥＬＩＳＰＯＴアッセイにて評価した。 Example 2
Peripheral blood mononuclear cells (PBMC) were separated from 50 ml of peripheral blood of HLA-A24-positive lung cancer patients or HLA-A24-positive healthy subjects who obtained CTL-derived informed consent by centrifugation in a Ficoll-Conlay density gradient. It was collected. Subsequently, CD8 MACS beads were used to separate CD8 positive lymphocytes and CD8 negative lymphocytes from PBMC.
In a 96-well plate, 200000 CD8 negative lymphocytes and the above HLA-A24-binding SOX2 peptide were mixed, allowed to stand at room temperature for 2 hours, and then subjected to radiation treatment for 100 Gy. Treated cells were co-cultured with 20 U / ml IL-2 (Takeda Pharmaceutical Co., Ltd.), 100,000 cells / well of CD8 positive lymphocytes, stimulated with CD8 positive lymphocytes once a week, and stimulated 3 times in total. Induced. The peptide-specific reactivity of the induced CTL was evaluated by cytotoxicity assay or ELISPOT assay.
実施例３
細胞傷害アッセイ
Ｔ２Ａ２４にＣｒ５１を加え、ＲＰＭＩ培地中で、３７℃で１時間培養して放射線標識した後、ＲＰＭＩ培地で4回洗浄した。Ｃｒ標識したＴ２Ａ２４にＳＯＸ２ペプチドあるいはコントロールペプチド（ＨＩＶペプチド）を混ぜ、室温で１時間静置した。ＳＯＸ２ペプチドあるいはコントロールペプチドをパルスした２０００個／ウェルのＴ２Ａ２４と、実施例２にて誘導した１４０００個／ウェルのＣＤ８陽性リンパ球をＲＰＭＩ培地中で、３７℃で４時間共培養した。その後上清を回収し、ガンマカウンターにてガンマ線量を測定した。 Example 3
Cr 51 was added to the cytotoxicity assay T2A24, cultured for 1 hour at 37 ° C. in RPMI medium, radiolabeled, and then washed four times with 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. 2000 / well T2A24 pulsed with SOX2 peptide or control peptide and 14000 / well CD8 positive lymphocytes induced in Example 2 were co-cultured in RPMI medium at 37 ° C. for 4 hours. Thereafter, the supernatant was collected, and the gamma dose was measured with a gamma counter.
細胞傷害アッセイ
Ｔ２Ａ２４にＣｒ５１を加え、ＲＰＭＩ培地中で、３７℃で１時間培養して放射線標識した後、ＲＰＭＩ培地で4回洗浄した。Ｃｒ標識したＴ２Ａ２４にＳＯＸ２ペプチドあるいはコントロールペプチド（ＨＩＶペプチド）を混ぜ、室温で１時間静置した。ＳＯＸ２ペプチドあるいはコントロールペプチドをパルスした２０００個／ウェルのＴ２Ａ２４と、実施例２にて誘導した１４０００個／ウェルのＣＤ８陽性リンパ球をＲＰＭＩ培地中で、３７℃で４時間共培養した。その後上清を回収し、ガンマカウンターにてガンマ線量を測定した。 Example 3
Cr 51 was added to the cytotoxicity assay T2A24, cultured for 1 hour at 37 ° C. in RPMI medium, radiolabeled, and then washed four times with 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. 2000 / well T2A24 pulsed with SOX2 peptide or control peptide and 14000 / well CD8 positive lymphocytes induced in Example 2 were co-cultured in RPMI medium at 37 ° C. for 4 hours. Thereafter, the supernatant was collected, and the gamma dose was measured with a gamma counter.
またＲＰＭＩ培地のみとペプチドパルスＴ２Ａ２４を培養した場合の線量を自然放出（ＳＰＲ）、細胞溶解液(２％のＮＰ４０)とペプチドパルスＴ２Ａ２４を培養した場合の線量を最大放出(ＭＸＲ)とし、細胞障害活性を(測定値－ＳＰＲ）／（ＭＸＲ－ＳＰＲ）×１００として算出した。
その結果、ＳＯＸ２＿１０９ペプチドをパルスしたＴ２Ａ２４に対して特異的に障害するＣＴＬを検出した。結果を図２に示す。コントロールペプチドであるＨＩＶ由来ペプチドでは、傷害細胞由来の放射線がほとんど検出されないのに対して、ＳＯＸ２＿１０９ペプチドでは多く検出された。 In addition, the dose when cultivating peptide pulse T2A24 only with RPMI medium is spontaneously released (SPR), and the dose when cultivating cell lysate (2% NP40) and peptide pulse T2A24 is maximum released (MXR). The activity was calculated as (measured value−SPR) / (MXR−SPR) × 100.
As a result, CTLs that specifically impaired T2A24 pulsed with SOX2 — 109 peptide were detected. The results are shown in FIG. In the HIV-derived peptide that is a control peptide, radiation derived from damaged cells was hardly detected, whereas in the SOX2_109 peptide, a large amount was detected.
その結果、ＳＯＸ２＿１０９ペプチドをパルスしたＴ２Ａ２４に対して特異的に障害するＣＴＬを検出した。結果を図２に示す。コントロールペプチドであるＨＩＶ由来ペプチドでは、傷害細胞由来の放射線がほとんど検出されないのに対して、ＳＯＸ２＿１０９ペプチドでは多く検出された。 In addition, the dose when cultivating peptide pulse T2A24 only with RPMI medium is spontaneously released (SPR), and the dose when cultivating cell lysate (2% NP40) and peptide pulse T2A24 is maximum released (MXR). The activity was calculated as (measured value−SPR) / (MXR−SPR) × 100.
As a result, CTLs that specifically impaired T2A24 pulsed with SOX2 — 109 peptide were detected. The results are shown in FIG. In the HIV-derived peptide that is a control peptide, radiation derived from damaged cells was hardly detected, whereas in the SOX2_109 peptide, a large amount was detected.
実施例４
ＥＬＩＳＰＯＴアッセイ
実験はHuman IFNγ ELISPOT set (BD)を使用して行った。ＥＬＩＳＰＯＴプレートに抗ＩＦＮγ抗体を４℃で一晩静置してコートした。Ｔ２Ａ２４にＳＯＸ２ペプチドあるいはコントロールペプチド(ＨＩＶペプチド)をそれぞれ加え、室温１時間静置した。５００００個のペプチドパルスしたＴ２Ａ２４と、実施例２にて誘導した１００００個のＣＴＬを抗ＩＦＮγ抗体をコートしたプレートで、３７℃で一晩共培養した。１×ＰＢＳと０．０５％のＴｗｅｅｎ２０で洗浄後、ビオチン標識抗ＩＦＮγ抗体を加え、室温で２時間静置した。１×ＰＢＳと０．０５％のＴｗｅｅｎ２０で洗浄後、ＨＲＰ標識ストレプトアビジンを加え、室温で１時間静置した。１×ＰＢＳと０．０５％のＴｗｅｅｎ２０で洗浄後、発色試薬を加え、スポット数を測定した。 Example 4
ELISPOT assay experiments were performed using a Human IFNγ ELISPOT set (BD). The ELISPOT plate was coated with the anti-IFNγ antibody by standing overnight at 4 ° C. SOX2 peptide or control peptide (HIV peptide) was added to T2A24, and allowed to stand at room temperature for 1 hour. 50000 peptide-pulsed T2A24 and 10000 CTL derived in Example 2 were co-cultured overnight at 37 ° C. on a plate coated with anti-IFNγ antibody. After washing with 1 × PBS and 0.05% Tween 20, a biotin-labeled anti-IFNγ antibody was added and allowed to stand at room temperature for 2 hours. After washing with 1 × PBS and 0.05% Tween 20, HRP-labeled streptavidin was added and allowed to stand at room temperature for 1 hour. After washing with 1 × PBS and 0.05% Tween 20, a coloring reagent was added and the number of spots was measured.
ＥＬＩＳＰＯＴアッセイ
実験はHuman IFNγ ELISPOT set (BD)を使用して行った。ＥＬＩＳＰＯＴプレートに抗ＩＦＮγ抗体を４℃で一晩静置してコートした。Ｔ２Ａ２４にＳＯＸ２ペプチドあるいはコントロールペプチド(ＨＩＶペプチド)をそれぞれ加え、室温１時間静置した。５００００個のペプチドパルスしたＴ２Ａ２４と、実施例２にて誘導した１００００個のＣＴＬを抗ＩＦＮγ抗体をコートしたプレートで、３７℃で一晩共培養した。１×ＰＢＳと０．０５％のＴｗｅｅｎ２０で洗浄後、ビオチン標識抗ＩＦＮγ抗体を加え、室温で２時間静置した。１×ＰＢＳと０．０５％のＴｗｅｅｎ２０で洗浄後、ＨＲＰ標識ストレプトアビジンを加え、室温で１時間静置した。１×ＰＢＳと０．０５％のＴｗｅｅｎ２０で洗浄後、発色試薬を加え、スポット数を測定した。 Example 4
ELISPOT assay experiments were performed using a Human IFNγ ELISPOT set (BD). The ELISPOT plate was coated with the anti-IFNγ antibody by standing overnight at 4 ° C. SOX2 peptide or control peptide (HIV peptide) was added to T2A24, and allowed to stand at room temperature for 1 hour. 50000 peptide-pulsed T2A24 and 10000 CTL derived in Example 2 were co-cultured overnight at 37 ° C. on a plate coated with anti-IFNγ antibody. After washing with 1 × PBS and 0.05
結果を図３に示す。ＳＯＸ２＿１０９ペプチドをパルスしたＴ２Ａ２４に対して特異的に反応するＣＴＬを検出した。コントロールペプチドであるＨＩＶ由来ペプチドでは、Ｔ細胞からのＩＦＮγの放出が検出されなかったが、ＳＯＸ２＿１０９ペプチドでは多くのＩＦＮγが放出されたことが確認できる。
The results are shown in FIG. CTL specifically reacting with T2A24 pulsed with SOX2 — 109 peptide was detected. In the HIV-derived peptide that is a control peptide, release of IFNγ from T cells was not detected, but it can be confirmed that a large amount of IFNγ was released in the SOX2 — 109 peptide.
実施例５
各ＳＯＸ２由来ペプチドの細胞傷害性Ｔ細胞誘導能比較
ＨＬＡ－Ａ２４０２陽性の肺がん患者６人由来の末梢血を使用した。全ての患者からインフォームドコンセントを取得している。ＰＢＭＣｓを、Lymphoprep（登録商標）（Nycomed社、オスロ、ノルウェー）で、常法の密度勾配遠心分離法により分離した。 Example 5
Comparison of cytotoxic T cell induction ability of each SOX2-derived peptide Peripheral blood derived from 6 HLA-A2402-positive lung cancer patients was used. Informed consent is obtained from all patients. PBMCs were separated by conventional density gradient centrifugation with Lymphoprep® (Nycomed, Oslo, Norway).
各ＳＯＸ２由来ペプチドの細胞傷害性Ｔ細胞誘導能比較
ＨＬＡ－Ａ２４０２陽性の肺がん患者６人由来の末梢血を使用した。全ての患者からインフォームドコンセントを取得している。ＰＢＭＣｓを、Lymphoprep（登録商標）（Nycomed社、オスロ、ノルウェー）で、常法の密度勾配遠心分離法により分離した。 Example 5
Comparison of cytotoxic T cell induction ability of each SOX2-derived peptide Peripheral blood derived from 6 HLA-A2402-positive lung cancer patients was used. Informed consent is obtained from all patients. PBMCs were separated by conventional density gradient centrifugation with Lymphoprep® (Nycomed, Oslo, Norway).
（１）ＣＴＬ誘導
上記で分離したＰＢＭＣｓを、１×１０７ＰＢＭＣｓ／ウェルとなるように播種し、１０％のプールヒトＡＢ血清を含むＡＩＭ培地（ライフテクノロジーズ社）で培養した。ｐｃＤＮＡ３．１ ｐｌａｓｍｉｄ（Invitrogen）にＳＯＸ２全長をコードするｃＤＮＡを挿入し、これをマンノース被覆リポソームに包埋した（ＯＭＬ－ＳＯＸ２、株式会社バイオメッドコアにて作製）。１日目に、ＯＭＬ－ＳＯＸ２を０．１μｇ／ｍＬまたは１μｇ／ｍＬの濃度でパルスし、５０ＵのＩＬ－２を添加して１週間培養した。その後エンザイムリンクトエリスポット（以下、「ＥＬＩＳＰＯＴ」という）アッセイによりＳＯＸ２由来ペプチド特異的ＣＴＬｓを検出した。 (1) CTL induction The PBMCs separated as described above were seeded so as to be 1 × 10 7 PBMCs / well, and cultured in AIM medium (Life Technologies) containing 10% pooled human AB serum. The cDNA encoding the full length of SOX2 was inserted into pcDNA3.1 plasmid (Invitrogen) and embedded in mannose-coated liposomes (produced by OML-SOX2, Biomedcore Inc.). On the first day, OML-SOX2 was pulsed at a concentration of 0.1 μg / mL or 1 μg / mL, and 50 U of IL-2 was added and cultured for 1 week. Thereafter, SOX2-derived peptide-specific CTLs were detected by an enzyme linked eryspot (hereinafter referred to as “ELISPOT”) assay.
上記で分離したＰＢＭＣｓを、１×１０７ＰＢＭＣｓ／ウェルとなるように播種し、１０％のプールヒトＡＢ血清を含むＡＩＭ培地（ライフテクノロジーズ社）で培養した。ｐｃＤＮＡ３．１ ｐｌａｓｍｉｄ（Invitrogen）にＳＯＸ２全長をコードするｃＤＮＡを挿入し、これをマンノース被覆リポソームに包埋した（ＯＭＬ－ＳＯＸ２、株式会社バイオメッドコアにて作製）。１日目に、ＯＭＬ－ＳＯＸ２を０．１μｇ／ｍＬまたは１μｇ／ｍＬの濃度でパルスし、５０ＵのＩＬ－２を添加して１週間培養した。その後エンザイムリンクトエリスポット（以下、「ＥＬＩＳＰＯＴ」という）アッセイによりＳＯＸ２由来ペプチド特異的ＣＴＬｓを検出した。 (1) CTL induction The PBMCs separated as described above were seeded so as to be 1 × 10 7 PBMCs / well, and cultured in AIM medium (Life Technologies) containing 10% pooled human AB serum. The cDNA encoding the full length of SOX2 was inserted into pcDNA3.1 plasmid (Invitrogen) and embedded in mannose-coated liposomes (produced by OML-SOX2, Biomedcore Inc.). On the first day, OML-SOX2 was pulsed at a concentration of 0.1 μg / mL or 1 μg / mL, and 50 U of IL-2 was added and cultured for 1 week. Thereafter, SOX2-derived peptide-specific CTLs were detected by an enzyme linked eryspot (hereinafter referred to as “ELISPOT”) assay.
（２）ＥＬＩＳＰＯＴアッセイ
前記ＳＯＸ２由来ペプチドに対するＣＴＬｓの特異性を、既に報告されている方法（Tsuruma, Tら、J. Transl. Med.，6:24(2008)）に従って、ＩＦＮγ ＥＬＩＳＰＯＴアッセイにより判定した。まずマルチスクリーン９６ウェルプレート（ミリポア社、ベッドフォード、マサチューセッツ州）に、ＰＢＳ中の５μｇ／ｍＬの抗ＩＦＮγ捕捉抗体（Beckton Dickinson Biosciences）を１００μＬ／ウェルで添加し、４℃で一晩無菌的に静置してコートした。プレートを２００μＬ／ウェルで一回洗浄し、１０％ヒト血清入りＡＩＭ－Ｖ培地２００μＬ／ウェルを用いて室温で２時間ブロッキングした。抗原提示細胞として、ＨＬＡ－Ａ２４を発現するＣ１Ｒリンパ腫の亜細胞株Ｃ１Ｒ－Ａ２４細胞を１０μｇ／ｍＬの前記各ＳＯＸ２ペプチドおよびネガティブコントロールとしてｓｕｒｖｉｖｉｎ２Ｂペプチドでパルスしたものを用いた。その後、５×１０３個のＣＴＬｓを、５×１０４個の抗原提示細胞Ｃ１Ｒ－Ａ２４細胞と共培養した。３７℃、５％ＣＯ２で２４時間培養後、ＰＢＳでウェルを５回洗浄し、ビオチン化抗ヒトＩＦＮ－γ抗体および西洋ワサビペルオキシダーゼ結合アビジンとともにインキュベートした。形成されたＩＦＮγスポットをKS ELISPOT（Carl Zeiss）を用いてカウントした。 (2) ELISPOT assay The specificity of CTLs for the SOX2-derived peptide was determined by IFNγ ELISPOT assay according to a previously reported method (Tsuruma, T et al., J. Transl. Med., 6:24 (2008)). . First, 5 μg / mL anti-IFNγ capture antibody (Beckton Dickinson Biosciences) in PBS was added at 100 μL / well to a multiscreen 96-well plate (Millipore, Bedford, Mass.) And aseptically overnight at 4 ° C. Allowed to coat. The plate was washed once with 200 μL / well and blocked with 200 μL / well of AIM-V medium with 10% human serum for 2 hours at room temperature. As antigen-presenting cells, C1R lymphoma subcell line C1R-A24 cells expressing HLA-A24 were pulsed with 10 μg / mL of each of the SOX2 peptides and survivin2B peptide as a negative control. Thereafter, 5 × 10 3 CTLs were co-cultured with 5 × 10 4 antigen-presenting cells C1R-A24 cells. After 24 hours incubation at 37 ° C., 5% CO 2 , the wells were washed 5 times with PBS and incubated with biotinylated anti-human IFN-γ antibody and horseradish peroxidase-conjugated avidin. The formed IFNγ spots were counted using KS ELISPOT (Carl Zeiss).
前記ＳＯＸ２由来ペプチドに対するＣＴＬｓの特異性を、既に報告されている方法（Tsuruma, Tら、J. Transl. Med.，6:24(2008)）に従って、ＩＦＮγ ＥＬＩＳＰＯＴアッセイにより判定した。まずマルチスクリーン９６ウェルプレート（ミリポア社、ベッドフォード、マサチューセッツ州）に、ＰＢＳ中の５μｇ／ｍＬの抗ＩＦＮγ捕捉抗体（Beckton Dickinson Biosciences）を１００μＬ／ウェルで添加し、４℃で一晩無菌的に静置してコートした。プレートを２００μＬ／ウェルで一回洗浄し、１０％ヒト血清入りＡＩＭ－Ｖ培地２００μＬ／ウェルを用いて室温で２時間ブロッキングした。抗原提示細胞として、ＨＬＡ－Ａ２４を発現するＣ１Ｒリンパ腫の亜細胞株Ｃ１Ｒ－Ａ２４細胞を１０μｇ／ｍＬの前記各ＳＯＸ２ペプチドおよびネガティブコントロールとしてｓｕｒｖｉｖｉｎ２Ｂペプチドでパルスしたものを用いた。その後、５×１０３個のＣＴＬｓを、５×１０４個の抗原提示細胞Ｃ１Ｒ－Ａ２４細胞と共培養した。３７℃、５％ＣＯ２で２４時間培養後、ＰＢＳでウェルを５回洗浄し、ビオチン化抗ヒトＩＦＮ－γ抗体および西洋ワサビペルオキシダーゼ結合アビジンとともにインキュベートした。形成されたＩＦＮγスポットをKS ELISPOT（Carl Zeiss）を用いてカウントした。 (2) ELISPOT assay The specificity of CTLs for the SOX2-derived peptide was determined by IFNγ ELISPOT assay according to a previously reported method (Tsuruma, T et al., J. Transl. Med., 6:24 (2008)). . First, 5 μg / mL anti-IFNγ capture antibody (Beckton Dickinson Biosciences) in PBS was added at 100 μL / well to a multiscreen 96-well plate (Millipore, Bedford, Mass.) And aseptically overnight at 4 ° C. Allowed to coat. The plate was washed once with 200 μL / well and blocked with 200 μL / well of AIM-V medium with 10% human serum for 2 hours at room temperature. As antigen-presenting cells, C1R lymphoma subcell line C1R-A24 cells expressing HLA-A24 were pulsed with 10 μg / mL of each of the SOX2 peptides and survivin2B peptide as a negative control. Thereafter, 5 × 10 3 CTLs were co-cultured with 5 × 10 4 antigen-presenting cells C1R-A24 cells. After 24 hours incubation at 37 ° C., 5% CO 2 , the wells were washed 5 times with PBS and incubated with biotinylated anti-human IFN-γ antibody and horseradish peroxidase-conjugated avidin. The formed IFNγ spots were counted using KS ELISPOT (Carl Zeiss).
結果を図４に示す。表中の数字は計測されたスポット数を表している。ネガティブコントロールであるｓｕｒ２Ｂおよび（－）のいずれか数値の高い方の１．３倍以上のスポット数が計測されたものを陽性と判定した。表の下には各ペプチドに対して陽性を示した患者数を表している。ＳＯＸ２＿５０および＿５８に反応するＣＴＬはどの患者からも誘導されず、最も抗原性が低いペプチドであると考えられる。それに対し、ＳＯＸ２＿１２４および＿２１６に反応するＣＴＬは４人の患者から誘導され、最も抗原性の高いペプチドであると考えられる。
The results are shown in FIG. The numbers in the table represent the number of spots measured. A negative control of sur2B and (−) in which the number of spots that was 1.3 times higher than the higher one was measured was determined as positive. Below the table is the number of patients positive for each peptide. 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.
実施例６
細胞傷害アッセイ
（１）ＳＯＸ２由来ペプチドを抗原提示する細胞に対する傷害性
実施例５における患者Ａの末梢血リンパ球からＣＤ８陽性Ｔ細胞を実施例２と同様に分離し、その他の単核球細胞にはフィトヘマグルチニン（ＰＨＡ）を添加して芽球化させた。芽球化させた単核球細胞に放射線を照射し、前記ＳＯＸ２由来ペプチド１３種の混合物を１０μｇ／ｍＬの濃度で添加し、ＩＬ－２を５０単位添加して、ＣＤ８陽性Ｔ細胞とともに混合培養した。同様のペプチド刺激を１週間毎に３回行い、３回目の刺激から５日後に、Ｔ２－Ａ２４細胞、ＳＯＸ２ペプチド１３種の混合物を添加したＴ２－Ａ２４細胞（Ｔ２－Ａ２４－ＳＯＸ２）およびネガティブコントロールとしてヒト赤芽球様白血病細胞（Ｋ５６２）を標的として細胞傷害アッセイを行った。 Example 6
Cytotoxicity assay (1) Toxicity to cells presenting antigens of SOX2-derived peptides CD8 positive T cells were isolated from peripheral blood lymphocytes of patient A in Example 5 in the same manner as in Example 2, and other mononuclear cells were separated. Added phytohemagglutinin (PHA) and allowed to blast. Irradiate blasted mononuclear cells, add a mixture of 13 SOX2-derived peptides at a concentration of 10 μg / mL, add 50 units of IL-2, and mix culture with CD8 positive T cells did. Similar peptide stimulation was performed 3 times per week, and 5 days after the third stimulation, T2-A24 cells, T2-A24 cells (T2-A24-SOX2) added with a mixture of 13 SOX2 peptides, and negative control A cytotoxic assay was conducted targeting human erythroblast-like leukemia cells (K562).
細胞傷害アッセイ
（１）ＳＯＸ２由来ペプチドを抗原提示する細胞に対する傷害性
実施例５における患者Ａの末梢血リンパ球からＣＤ８陽性Ｔ細胞を実施例２と同様に分離し、その他の単核球細胞にはフィトヘマグルチニン（ＰＨＡ）を添加して芽球化させた。芽球化させた単核球細胞に放射線を照射し、前記ＳＯＸ２由来ペプチド１３種の混合物を１０μｇ／ｍＬの濃度で添加し、ＩＬ－２を５０単位添加して、ＣＤ８陽性Ｔ細胞とともに混合培養した。同様のペプチド刺激を１週間毎に３回行い、３回目の刺激から５日後に、Ｔ２－Ａ２４細胞、ＳＯＸ２ペプチド１３種の混合物を添加したＴ２－Ａ２４細胞（Ｔ２－Ａ２４－ＳＯＸ２）およびネガティブコントロールとしてヒト赤芽球様白血病細胞（Ｋ５６２）を標的として細胞傷害アッセイを行った。 Example 6
Cytotoxicity assay (1) Toxicity to cells presenting antigens of SOX2-derived peptides CD8 positive T cells were isolated from peripheral blood lymphocytes of patient A in Example 5 in the same manner as in Example 2, and other mononuclear cells were separated. Added phytohemagglutinin (PHA) and allowed to blast. Irradiate blasted mononuclear cells, add a mixture of 13 SOX2-derived peptides at a concentration of 10 μg / mL, add 50 units of IL-2, and mix culture with CD8 positive T cells did. Similar peptide stimulation was performed 3 times per week, and 5 days after the third stimulation, T2-A24 cells, T2-A24 cells (T2-A24-SOX2) added with a mixture of 13 SOX2 peptides, and negative control A cytotoxic assay was conducted targeting human erythroblast-like leukemia cells (K562).
細胞傷害アッセイによる細胞傷害性計測は、５１Ｃｒ放出アッセイによって行った。標的細胞を１００μＣｉの５１Ｃｒで、３７℃で１時間標識し、ＲＰＭＩ１６４０培地で３回洗浄した。その後５１Ｃｒで標識した標的細胞を、エフェクター細胞とともに、様々なエフェクター／標的比率（Ｅ／Ｔ率）で、３７℃で６時間、Ｖ底の９６ウェルマイクロタイタープレートでインキュベートした。その後、上清を採集し、ガンマカウンターで放射能を計測した。％特異的細胞溶解（細胞傷害活性）は、実施例３と同様に計算した。
結果を図５ａ）に示す。ＳＯＸ２由来ペプチドと混合培養したＣＤ８陽性Ｔ細胞は、ＳＯＸ２由来ペプチドを添加したＴ２－Ａ２４細胞を特異的に傷害した。 Cytotoxicity measurement by cytotoxicity assays were performed by a 51 Cr release assay. Target cells were labeled with 100 μCi 51 Cr for 1 hour at 37 ° C. and washed 3 times with RPMI 1640 medium. Target cells labeled with 51 Cr were then incubated with effector cells at various effector / target ratios (E / T ratio) at 37 ° C. for 6 hours in a V-bottom 96-well microtiter plate. Thereafter, the supernatant was collected and the radioactivity was measured with a gamma counter. % Specific cell lysis (cytotoxic activity) was calculated as in Example 3.
The results are shown in FIG. CD8 positive T cells mixed and cultured with SOX2-derived peptides specifically damaged T2-A24 cells to which SOX2-derived peptides were added.
結果を図５ａ）に示す。ＳＯＸ２由来ペプチドと混合培養したＣＤ８陽性Ｔ細胞は、ＳＯＸ２由来ペプチドを添加したＴ２－Ａ２４細胞を特異的に傷害した。 Cytotoxicity measurement by cytotoxicity assays were performed by a 51 Cr release assay. Target cells were labeled with 100 μCi 51 Cr for 1 hour at 37 ° C. and washed 3 times with RPMI 1640 medium. Target cells labeled with 51 Cr were then incubated with effector cells at various effector / target ratios (E / T ratio) at 37 ° C. for 6 hours in a V-bottom 96-well microtiter plate. Thereafter, the supernatant was collected and the radioactivity was measured with a gamma counter. % Specific cell lysis (cytotoxic activity) was calculated as in Example 3.
The results are shown in FIG. CD8 positive T cells mixed and cultured with SOX2-derived peptides specifically damaged T2-A24 cells to which SOX2-derived peptides were added.
（２）ＳＯＸ２発現細胞に対する傷害性
（１）と同様に、ＨＬＡ－Ａ２４陽性ＬＨＫ２肺がん細胞およびＳＯＸ２遺伝子導入ＬＨＫ２細胞（ＬＨＫ２－ＳＯＸ２）を標的として、細胞傷害アッセイを行った。
結果を図５ｂ）に示す。ＳＯＸ２由来ペプチドと混合培養したＣＤ８陽性Ｔ細胞は、ＳＯＸ２を強制発現したＬＨＫ２細胞に対して高い細胞傷害活性を示した。 (2) Toxicity to SOX2-expressing cells In the same manner as in (1), cytotoxicity assays were performed targeting HLA-A24-positive LHK2 lung cancer cells and SOX2 gene-introduced LHK2 cells (LHK2-SOX2).
The results are shown in FIG. CD8 positive T cells co-cultured with SOX2-derived peptides showed high cytotoxic activity against LHK2 cells forcibly expressing SOX2.
（１）と同様に、ＨＬＡ－Ａ２４陽性ＬＨＫ２肺がん細胞およびＳＯＸ２遺伝子導入ＬＨＫ２細胞（ＬＨＫ２－ＳＯＸ２）を標的として、細胞傷害アッセイを行った。
結果を図５ｂ）に示す。ＳＯＸ２由来ペプチドと混合培養したＣＤ８陽性Ｔ細胞は、ＳＯＸ２を強制発現したＬＨＫ２細胞に対して高い細胞傷害活性を示した。 (2) Toxicity to SOX2-expressing cells In the same manner as in (1), cytotoxicity assays were performed targeting HLA-A24-positive LHK2 lung cancer cells and SOX2 gene-introduced LHK2 cells (LHK2-SOX2).
The results are shown in FIG. CD8 positive T cells co-cultured with SOX2-derived peptides showed high cytotoxic activity against LHK2 cells forcibly expressing SOX2.
本発明に基づく癌ワクチンまたは抗癌剤は、従来の治療法では治療や予防することが困難であった癌に対する有効な治療または予防手段となりうる。本発明は医療の向上、人類の福祉に貢献する。
The cancer vaccine or anticancer agent based on the present invention can be an effective treatment or prevention means for cancer that has been difficult to treat or prevent by conventional treatment methods. The present invention contributes to medical improvement and human welfare.
Claims (9)
- 癌細胞を標的とする細胞傷害性Ｔ細胞を誘導しうるペプチドであって、該ペプチドが、Ｓｏｘ２遺伝子によってコードされるポリペプチド由来のアミノ酸配列からなるか、またはそのアミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されているアミノ酸配列からなり、かつＨＬＡ－Ａ２４によって抗原提示される、前記ペプチド。 A peptide capable of inducing cytotoxic T cells targeting cancer cells, the peptide consisting of 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, which consists of an amino acid sequence in which amino acids are deleted, substituted or added, and is presented as an antigen by HLA-A24.
- ペプチドが、配列番号１、４～９および１２に示されるアミノ酸配列、または該アミノ酸配列において１もしくは数個のアミノ酸が欠失、置換もしくは付加されている配列からなる、請求項１に記載のペプチド。 The peptide according to claim 1, wherein the peptide consists of the amino acid sequence shown in 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. .
- 請求項１または２に記載のペプチドにより誘導された、細胞傷害性Ｔ細胞。 Cytotoxic T cells induced by the peptide according to claim 1 or 2.
- 請求項１または２に記載のペプチドをコードする、ＤＮＡ。 DNA which encodes the peptide according to claim 1 or 2.
- 請求項１もしくは２に記載のペプチドおよび／または請求項３に記載の細胞傷害性Ｔ細胞および／または請求項４に記載のＤＮＡを含む、医薬組成物。 A pharmaceutical composition comprising the peptide according to claim 1 or 2 and / or the cytotoxic T cell according to claim 3 and / or the DNA according to claim 4.
- 医薬組成物が、癌ワクチンである、請求項５に記載の医薬組成物。 The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is a cancer vaccine.
- 医薬組成物が、抗癌剤である、請求項５に記載の医薬組成物。 The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is an anticancer agent.
- 医薬組成物が、ＤＮＡワクチンである、請求項５に記載の医薬組成物。 The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is a DNA vaccine.
- 癌細胞を標的とする細胞傷害性Ｔ細胞を誘導するための、請求項１または２に記載のペプチドの使用。 Use of the peptide according to claim 1 or 2 for inducing cytotoxic T cells targeting cancer cells.
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| JP2020531846A (en) * | 2017-08-21 | 2020-11-05 | サヴィセル ダイアグノスティック リミテッドＳａｖｉｃｅｌｌ Ｄｉａｇｎｏｓｔｉｃ Ｌｔｄ． | How to Diagnose and Treat Lung Cancer |
| CN113861276A (en) * | 2021-10-22 | 2021-12-31 | 厦门大学 | Polypeptide for targeted binding of binding domain in Sox2-CDP protein complex on CDP as well as synthetic method and application thereof |
| WO2022066965A3 (en) * | 2020-09-24 | 2022-07-07 | Fred Hutchinson Cancer Research Center | Immunotherapy targeting sox2 antigens |
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| WO2017200017A1 (en) * | 2016-05-19 | 2017-11-23 | 東洋製罐グループホールディングス株式会社 | Cell preparation method, and cell culture vessel |
| JP2020531846A (en) * | 2017-08-21 | 2020-11-05 | サヴィセル ダイアグノスティック リミテッドＳａｖｉｃｅｌｌ Ｄｉａｇｎｏｓｔｉｃ Ｌｔｄ． | How to Diagnose and Treat Lung Cancer |
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