JP4547197B2 - Cancer specific tumor antigen - Google Patents

Description

  The present invention relates to cancer-specific tumor antigen peptides and the use of the antigen peptides in cancer vaccine therapy.

  In recent years, it has become clear that cytotoxic T lymphocytes responsible for cellular immunity play a major role in tumor rejection in vivo. Specifically, the tumor is eliminated by the cytotoxic T lymphocytes recognizing a tumor antigen presented on the tumor cell and killing the tumor cell.

  As a tumor antigen, Boon T. et al. Of the Ludwig Cancer Research Institute in Belgium in 1991 used a cDNA expression cloning method using autologous tumor-specific cytotoxic T lymphocytes as a probe to detect tumor rejection of malignant melanoma. The first MAGE gene encoding an antigen was identified (Non-patent Document 1). Further, in 1994, NIH Rosenberg S.A. et al. Identified MART-1, gp100 encoding a tumor antigen from malignant melanoma (Non-patent Documents 2 and 3). Since then, many genes encoding tumor antigens have been identified. For some of these tumor antigens, peptide epitopes presented within the major histocompatibility antigen (MHC) class I groove have also been identified and damage tumor cells in vitro by stimulation of such tumor antigen peptides It has also been reported that cytotoxic T lymphocytes can be induced (for example, Patent Documents 1 and 2). In addition, vaccine effects using these tumor antigen peptides have been partially reported in clinical patients mainly in patients with malignant melanoma.

  In general, in tumor rejection, a tumor antigen is first produced as a protein in a tumor cell, and then decomposed into a peptide of 8 to 11 amino acids to form an antigen peptide, which is a major histocompatibility antigen (MHC) human leukocyte antigen (HLA). ) Bound to the molecule and presented as a complex on the tumor cell surface. Cytotoxic T lymphocytes recognize the complex of antigen peptide and HLA molecule on the tumor cell and kill the tumor cell. The phenomenon in which cytotoxic T lymphocytes simultaneously recognize an HLA molecule bound to a tumor antigen peptide when recognizing the tumor antigen peptide is called HLA restriction. This HLA molecule is a cell membrane antigen expressed on almost all nucleated cells, and is roughly classified into a class I antigen and a class II antigen. Among these, it is a class I antigen that is presented on cells in a state of being bound to an antigenic peptide and recognized by cytotoxic T lymphocytes. HLA class I is further classified into HLA-A, HLA-B, and HLA-C. These HLA class I are known to be rich in diversity, for example, HLA-A includes HLA-A1, HLA-A2, HLA-A24, HLA-A34, HLA-B includes HLA-B8, HLA-C has polymorphisms such as HLA-Cw1, HLA-Cw3, and HLA-Cw6, such as HLA-B27, HLA-B46, and HLA-B53. Human individuals have these HLA class I genes in combinations of polymorphisms that vary from individual to individual. It has also been reported that the frequency of occurrence of this HLA class I gene within a population varies depending on the human population. For example, the HLA-A24 gene (HLA-A24 allele) is about 60% of the Japanese population, about 20% of whites, Approximately 12% of Africans hold it. Tumor antigen peptide (common antigen) capable of inducing cytotoxic T lymphocytes capable of damaging not only autologous tumor cells but also allogeneic tumor cells, and a tumor capable of binding to HLA molecules that appear frequently in the human population If an antigenic peptide is identified, it can be applied to many cases and is considered very useful.

  In lung cancer, it is difficult to establish a lung cancer cell line, and further induces cytotoxic T lymphocytes due to the fact that costimulatory molecules that activate lymphocytes are attenuated or lacked on the surface of tumor cells. It has been difficult to establish a stable experimental system that can be observed, and tumor antigens, particularly common antigens, that are considered useful for treatment have not been identified so far.

  Identification of tumor antigens as epitopes of cytotoxic T lymphocytes in cancer is extremely important in developing immunotherapy as cancer vaccine therapy.

International Publication No. 03/050140 Pamphlet JP-A-9-151200 Van der Bruggen, P et al., Science, (1991) 254, p.1643-1647 Kawakami, Y. et al., Proc. Natl. Acad. Sci. USA, (1994) 91, p.3515-3519 Kawakami, Y. et al., Proc. Natl. Acad. Sci. USA, (1994) 91, p.6458-6462

  The present invention relates to tumor antigen peptides that induce cytotoxic T lymphocytes (CTLs) that damage cancer cells in a HLA-A24-restricted manner, induced CTLs, and their use in cancer-specific vaccine therapy. The purpose is to provide.

As a result of diligent research to solve the above problems, the present inventors established a cytotoxic T lymphocyte clone that specifically recognizes and damages cancer cells restricted to HLA-A24, and the cytotoxicity thereof. A cDNA encoding a tumor antigen recognized by a sex T lymphocyte clone was isolated, and a novel antigen peptide consisting of a 9 amino acid sequence was identified as the tumor antigen. The present invention has been completed based on such a result, that is, as follows.
[1] A peptide consisting of the amino acid sequence of NYGFQIHTK (SEQ ID NO: 1).
[2] A DNA encoding the peptide according to [1] above.
[3] DNA having the base sequence represented by SEQ ID NO: 2.
[4] An expression vector comprising the DNA according to [2] or [3] above.
[5] A cytotoxic T lymphocyte that recognizes a peptide consisting of the amino acid sequence of NYGFQIHTK (SEQ ID NO: 1) in an HLA-A24-restricted manner. Particularly preferred cytotoxic T lymphocytes include cytotoxic T lymphocytes represented by accession number FERM P-19760.
[6] A peptide comprising an amino acid sequence of NYGFQIHTK (SEQ ID NO: 1), wherein the peptide comprises an amino acid sequence in which one or several amino acids are deleted, substituted and / or added, and the cell according to [5] above The peptide, which is recognized by HLA-A24 restricted by impaired T lymphocytes.
[7] A vaccine for treating or preventing cancer comprising the peptide according to [1] or [6] above.
[8] A vaccine for treating or preventing cancer comprising the expression vector according to [4] above.
[9] The vaccine according to [7] or [8] above, wherein the cancer is lung cancer.
[10] The vaccine according to [7] to [9] above, for a patient positive for HLA-A24.
[11] An antigen-presenting cell in which the peptide according to [1] or [6] is presented on an HLA-A24 molecule.
[12] A cell medicine containing the cytotoxic T lymphocyte according to the above [5].
[13] A cell medicine comprising the antigen-presenting cell according to [11] above.
[14] A method for obtaining cytotoxic T lymphocytes that damage HLA-A24-restricted cancer cells, comprising the following steps (1) to (3):
(1) HLA-, wherein the peptide according to [1] or [6] and an HLA-A24 positive cell are mixed and cultured, and the peptide according to [1] or [6] is presented on an HLA-A24 molecule Preparing A24 positive cells;
(2) A step of inducing cytotoxic T lymphocytes by co-culturing a cell population containing HLA-A24-positive cancer cells and a cell population containing HLA-A24-positive CTL precursor cells, and
(3) Cells obtained in step (1) of cytotoxic T lymphocytes by co-culturing the cells prepared in step (1) and the cytotoxic T lymphocytes induced in step (2) Measuring the cytotoxic activity against.
[15] A method for inducing cytotoxic T lymphocytes that damage HLA-A24-restricted cancer cells, comprising the following steps (1) and (2):
(1) A mixed culture of the peptide according to [1] or [6] above and an HLA-A24 positive cell having antigen-presenting ability, and the peptide according to [1] or [6] above as an HLA-A24 molecule Preparing an antigen-presenting cell presented in 1.
(2) A step of mixing and culturing the antigen-presenting cells prepared in step (1) and a cell population containing HLA-A24-positive CTL precursor cells.

  The cancer-specific tumor antigen peptide of the present invention is specific to cytotoxic T lymphocytes that specifically recognize and damage cancer cells in a HLA-A24-restricted state while being presented to HLA-A24 molecules on the cells. Recognized antigenic peptide. By using the cancer-specific tumor antigen peptide of the present invention, it is possible to specifically induce HLA-A24-restricted cytotoxic activity against cancer cells by cytotoxic T lymphocytes. Moreover, if this is utilized, it is also possible to specifically select cytotoxic T lymphocytes that damage cancer cells in an HLA-A24-restricted manner. The cancer-specific tumor antigen peptide of the present invention can also specifically induce cytotoxic T lymphocytes that damage cancer cells in a HLA-A24-restricted manner.

  The cytotoxic T lymphocytes that damage cancer cells in an HLA-A24-restricted manner of the present invention may specifically damage cancer cells by recognizing the tumor antigen peptide of the present invention in an HLA-A24-restricted manner. it can. This cytotoxic T lymphocyte of the present invention recognizes a tumor antigen peptide bound to HLA-A24, which is retained by about 60% of Japanese, and is not only an autologous cancer cell but also an allogeneic cancer cell. Can also be specifically impaired, and can be applied to a wide range of patients. The cytotoxic T lymphocytes of the present invention are also very useful in immunotherapy because they do not damage normal cells and can destroy cancer cells such as lung cancer cells with high efficiency and have few side effects. . In view of the fact that lung cancer is a cancer with a poor prognosis compared to other solid cancers, cytotoxic T lymphocytes having such excellent cancer cell damaging action are useful in the treatment and prevention of solid cancers including lung cancer. It is particularly useful.

  Furthermore, the DNA encoding the lung cancer-specific antigen peptide of the present invention can be used not only for producing the tumor antigen peptide of the present invention, but also incorporated into an expression vector and expressed in vitro or in vivo. Of tumor antigen peptides can be used to present antigens on cells.

  In the present specification, “(cell) impairs HLA-A24 restriction” means that a cytotoxic T lymphocyte is a complex of an HLA-A24 molecule expressed on a cell and an antigenic peptide bound thereto. Recognize and mean to kill that cell. Furthermore, “(cytotoxic T lymphocyte recognizes antigen peptide in an HLA-A24-restricted manner)” means that the cytotoxic T lymphocyte expresses the HLA-A24 molecule expressed on the cell and the antigen bound thereto. It means to specifically bind to a complex with a peptide via a T cell receptor (TCR) to proliferate and activate. The cytotoxic T lymphocyte recognizes the antigen peptide in an HLA-A24-restricted manner, indicating that the cytotoxic T lymphocyte exhibits cytotoxic activity against the target cell presenting the antigen peptide and reacts to the target cell. This can be confirmed by producing cytokines.

  As used herein, “inducing cytotoxic T lymphocytes” means that cytotoxic T lymphocytes activated by antigen stimulation of CTL precursor cells proliferate.

  In the present specification, “cancer” means a malignant tumor (carcinoma) composed of epithelial cells, a malignant tumor (sarcoma) composed of non-epithelial cells, and a malignant tumor including hematopoietic tumors. In general, most cancers other than hematopoietic tumors are also called solid tumors because they form a tumor mass. Representative examples of carcinoma include lung cancer, breast cancer, uterine cancer, ovarian cancer, stomach cancer, colon cancer, pharyngeal cancer, laryngeal cancer, tongue cancer and the like. Representative examples of sarcomas include osteosarcoma, chondrosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma and the like. Representative examples of hematopoietic tumors include leukemia, malignant lymphoma, myeloma and the like.

  In the present specification, “lung cancer” refers to a malignant tumor (eg, adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, large cell lung cancer, carcinoid, adenoid cystic cancer, mucoepidermoid carcinoma, lung, which occurs in the lung. Cell carcinoma, polymorphic cancer, etc.). “Lung cancer” in the present invention may be primary or metastatic.

  Hereinafter, the present invention will be described in more detail.

1. Tumor antigen peptide and DNA encoding tumor antigen peptide
In one embodiment, the tumor antigen peptide of the present invention is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (NYGFQIHTK). This tumor antigen peptide is a protein (tumor antigen) consisting of 617 amino acids encoded by the open reading frame in cDNA clone 214.2 (3339 bp, SEQ ID NO: 3), which is a splicing variant of the known gene Tara, expressed in lung cancer cells. And is encoded by the nucleotide sequence: AACTATGGCTTCCAGATCCACACCAAG (SEQ ID NO: 2). In the present invention, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 recognizes cytotoxic T lymphocytes that are restricted to HLA-A24 (for example, cytotoxic T lymphocyte clone F ₂ b / 5 described later). For the first time to be specifically recognized.

Furthermore, the present invention relates to a peptide comprising an amino acid sequence in which one or several (2 to 5) amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. Among these peptides, cytotoxic T lymphocytes that recognize the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in an HLA-A24-restricted manner (for example, cytotoxic T lymphocyte clone F ₂ b / 5 described later). Peptides recognized as HLA-A24 restricted are included in the tumor antigen peptides of the present invention.

  The tumor antigen peptide of the present invention can be produced by any peptide synthesis method. As peptide synthesis methods, for example, chemical synthesis techniques such as liquid phase peptide synthesis methods and solid phase peptide synthesis methods are often used. In solid phase peptide synthesis methods, synthesis using an automatic peptide synthesizer is also commonly used. (For example, “Seminar in Experimental Chemistry 1 Protein IV” (1992) edited by Japanese Biochemical Society, Tokyo Chemical Doujin; “The Peptides: Analysis, Synthesis, Biology” Vol. 1-5, ed. By E. Gross, J Meienhofer; Vol. 6-9, ed. By S. Udenfriend, J. Meienhofer, Academic Press, New York (1979-1987)). Alternatively, peptide synthesis may be performed by a recombinant method using DNA encoding the tumor antigen peptide of the present invention. For example, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is produced by incorporating DNA consisting of the base sequence shown in SEQ ID NO: 2 into an expression vector, introducing it into an expression host such as E. coli and culturing. Can do. The produced tumor antigen peptide is further purified by a conventional method, for example, gel chromatography, ion column chromatography, affinity chromatography, fractionation method based on solubility difference using ammonium sulfate, alcohol, etc., immunoadsorption method, etc. It can be purified and / or recovered.

  The tumor antigen peptide of the present invention is recognized by HLA-A24-restricted cells by cytotoxic T lymphocytes that specifically damage cancer cells in a HLA-A24-restricted manner. It can be used for the selection or induction of specifically damaged cytotoxic T lymphocytes.

  Furthermore, DNA encoding the tumor antigen peptide of the present invention (for example, DNA consisting of the base sequence represented by SEQ ID NO: 2) and a vector containing the DNA are also included in the scope of the present invention.

2. Induction of cytotoxic T lymphocytes The cytotoxic T lymphocytes that damage cancer cells in an HLA-A24-restricted manner according to the present invention can be induced in vitro by the following method.

(1) Induction by stimulation of T lymphocytes using HLA-A24 positive cancer cells The cytotoxic T lymphocytes of the present invention that specifically damage cancer cells in a restricted manner to HLA-A24 are HLA-A24 positive. It can be induced by stimulating CTL progenitor cells with cancer cells.

  The HLA-A24-positive cancer cells used in this method are not limited, and cancer cell lines established by conventional methods from tumors collected from HLA-A24-positive cancer patients can be used. Whether or not the tumor patient is “HLA-A24 positive” can be determined by any commonly used method including DNA typing methods such as serological methods and PCR-SSP (polymerase chain reaction-sequence specific primers) methods. What is necessary is just to judge by the HLA typing method. Various HLA typing reagents and HLA typing kits used for HLA typing are also commercially available, such as the HLA Typing series (Takara Bio) and the “Wakunaga MPH-2 HLA typing reagent” series (Yunaga Pharmaceutical).

  In order to further increase the induction efficiency of cytotoxic T lymphocytes, CD80 cDNA or CD86 cDNA, which is a costimulatory molecule, may be introduced into HLA-A24-positive cancer cells by, for example, lipofection. Cells introduced with the CD80 or CD86 gene have been reported to have the ability to induce T cell proliferation, cytokine production, and cytotoxic activity. The base sequences of CD80 cDNA and CD86 cDNA are registered in the DDBJ / EMBL / GenBank international base sequence database as accession numbers NP005182 and NP062261, respectively. HLA-A24-positive lung cancer cells into which CD80 or CD86 gene has been introduced are preferably inactivated by irradiation (100 Gy) before being used for lymphocyte stimulation.

  As the CTL progenitor cells to be stimulated (progenitor cells of cytotoxic T lymphocytes), a cell population containing CTL progenitor cells obtained from the same cancer patient from whom HLA-A24-positive cancer cells were collected may be used. . “Cell population containing CTL progenitor cells” means a cell population obtained from peripheral lymph nodes and blood, and usually, peripheral blood mononuclear cells (PBMC), regional lymph nodes, infiltrating lymphocytes in tumors Etc. are used.

  More specifically, stimulation of CTL progenitor cells using HLA-A24-positive cancer cells is performed by culturing HLA-A24-positive cancer cells and a cell population containing CTL progenitor cells in a mixed culture, and then with cancer cells. The stimulation by mixed culture may be performed by repeatedly applying to the cell population containing the CTL precursor cells. In one embodiment, HLA-A24-positive cancer cells are added to a cell population containing CTL precursor cells at a ratio of 10: 1 and mixed and cultured once a week for a total of 3 to 4 times. -A24-restricted cytotoxic T lymphocytes that damage cancer cells can be induced.

(2) Induction of cytotoxic T lymphocytes by stimulation of lymphocytes using tumor antigen peptides The cytotoxic T lymphocytes that inhibit cancer cells in a HLA-A24-restricted manner according to the present invention are the tumor antigens of the present invention. It can also be induced by stimulation of CTL precursor cells using a peptide, preferably a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. As a method for inducing cytotoxic T lymphocytes using the tumor antigen peptide of the present invention, any method known to those skilled in the art can be used. Methods known to those skilled in the art include, for example, the method of van der Bruggen et al. And the method of Hill et al. (Plebanski, M. et al., Eur. J. Immunol. (1994) 25, p. .1783; Tanaka, F. et al., Cancer Immunol. Immunother. (1997) 44, p.21), DC method using dendritic cells (Tsai, V. et al., J. Immunol. (1996) 158 , p. 1796).

  In general, cytotoxic T lymphocytes can be induced using the tumor antigen peptide of the present invention as follows. First, the tumor antigen peptide of the present invention is added (pulsed) to HLA-A24-positive cells that have antigen-presenting ability such as dendritic cells and macrophages, so that the tumor antigen peptide of the present invention is HLA- Prepare antigen-presenting cells presented on the A24 molecule. Next, by co-culturing a cell population containing CTL precursor cells with this antigen-presenting cell, cytotoxic T lymphocytes that specifically damage cancer cells in a restricted manner by HLA-A24 are induced.

  In one embodiment, first, plate-adherent cells in peripheral blood mononuclear cells (PBMC) derived from an HLA-A24-positive healthy person or cancer patient are treated with GM-CSF (1,000 U / ml), IL-4 (1,000 U / ml) in the presence of 7 days to induce dendritic cells (DC). The dendritic cells are pulsed with the tumor antigen peptide of the present invention (20 μg / ml) and then irradiated with X-rays (55 Gy) to prepare antigen presenting cells (stimulators). When DC cannot be used, the tumor antigen peptide (20 μg / ml) of the present invention is pulsed into peripheral blood mononuclear cells (PBMC) derived from the same HLA-A24 positive healthy subject or the same cancer patient, and then irradiated with X-rays ( You may use what performed 30 Gy). On the other hand, the wells of a 24-well plate using the same peripheral blood mononuclear cells (PBMC) from healthy individuals positive for HLA-A24, peripheral blood mononuclear cells (PBMC) from cancer patients, or regional lymph node lymphocytes (responder) In addition, in the presence of IL-2, IL-4, and IL-7, the ratio of stimulator and responder is 1:10. After 7 and 14 days from the start of the culture, the antigen-presenting cells obtained by pulsing the tumor antigen peptide of the present invention as described above were added to carry out restimulation, and IL-2 (30 U / ml every 2 to 3 days thereafter. ) And culture.

  As a medium for cell culture used for induction of cytotoxic T lymphocytes, any medium capable of surviving T lymphocytes may be used. For example, RHAMα medium (Kawai, K., Sasaki, T., Saijo-Kurita, K., Akaza, H., Koiso, K., and Ohno, T., Cancer Immunol. Immunother. 35, 225-229, 1992 LAK mcdium), AIMV medium (GIBCO BRL, Life Technologies, INC.), Or RPMI1640 medium with various cytokines such as interleukin-2 and fetal calf serum (FCS) added be able to.

  For example, a medium obtained by adding interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-7, etc. to RHAMα medium can be used. Although the concentration of these cytokines is not limited, for example, any of them may be used at about 1 U / ml to 2,000 U / ml. These cytokines may be combined and added simultaneously, or may be added alone.

Culture conditions may follow conditions well known to those skilled in the art. For example, the culture temperature is 33 ° C to 41 ° C, preferably 37 ° C. An inert gas containing air or an appropriate concentration of oxygen and an appropriate concentration of carbon dioxide gas (for example, 5% CO ₂ ) for maintaining the pH of the medium at about 7.4 can be used as the gas phase. The culture is preferably 4 to 10 days, more preferably 7 days. T lymphocyte cells that have been induced by such culture can be used as the cytotoxic T lymphocytes of the present invention.

  The cytotoxic T lymphocytes induced by the above method (1) or (2) have the ability to specifically damage cancer cells in an HLA-A24-restricted manner. Such cytotoxic T lymphocytes can be obtained as a clone from a cell population by the method described in Section 3 below. Cytotoxic T lymphocytes or cytotoxic T lymphocyte clones that specifically damage cancer cells in a restricted manner by HLA-A24 are included in the scope of the present invention.

  In addition, the antigen-presenting cell in which the above-described tumor antigen peptide of the present invention is presented on the HLA-A24 molecule is also included in the scope of the present invention.

3. Acquisition of cytotoxic T lymphocytes that specifically damage cancer cells in a restricted manner with HLA-A24 From the cytotoxic T lymphocytes (bulk CTL) induced as described in Section 2 above, the limiting dilution method was used. In this way, cytotoxic T lymphocyte clones can be obtained. With respect to this cytotoxic T lymphocyte clone, it is preferable to measure the cytotoxic activity against cancer cells and evaluate HLA-A24 restriction.

  Measurement of cytotoxic activity against cancer cells with respect to cytotoxic T lymphocytes may be performed using any cytotoxicity test known to those skilled in the art. For example, as a general cytotoxicity test, cytotoxic T lymphocytes and cancer cells derived from HLA-A24 positive patients (preferably those derived from the same patient as the cytotoxic T lymphocytes) are used in vitro. The degree of damage of cancer cells may be examined by contacting with This degree of cancer cell damage can be regarded as the cytotoxic activity of cytotoxic T lymphocyte clones. Although it does not limit as a cancer cell used for a cytotoxicity test, a lung cancer cell is preferable.

The degree of damage of cancer cells can be expressed by cell lysis (%) of cancer cells. The cell lysis (%) of cancer cells may be obtained by directly counting the number of cancer cells damaged by measurement under a microscope or the like. Preferably, the cell lysis (%) of the cancer cell is such that, for example, the target cell is pre-labeled with a radioisotope such as ⁵¹ Cr or other labeling substance and contacted with a cytotoxic T lymphocyte clone. By measuring the amount of labeled substance released into the cell, the number of damaged cancer cells can be calculated indirectly (Chromium release test; Takenoyama, M. et al., (2001) Jpn J. Cancer Res., 92, p. 309-315). Since DNA fragmentation is observed in damaged cells, the nuclear DNA of cancer cells is pre-labeled with a radioisotope such as [ ³ H] thymidine or [ ¹²⁵ I] uridine, and the amount of extracellular release is determined. The number of damaged cancer cells can also be indirectly calculated by measuring with radioactivity or measuring the degree of DNA fragmentation by DNA electrophoresis. Alternatively, by utilizing the fact that dyes such as trypan blue and nigrosine are easily taken up by damaged cells and remain without being excluded, these dyes are added to the cells, and the cells that have taken up the dyes are damaged. Can also be easily identified as an isolated cell (dye exclusion test).

  The cytotoxic activity may also be measured by a cytokine production test that measures the ability of cytokine production to the culture supernatant. Cytokine production tests that can be suitably used in the present invention include, for example, the method described in Hansen et al., (1989) J. Immunol. Methods, 119, p. 203-210, or Takenoyama, M. et al., ( 2001) Jpn. J. Cancer Res., 92, p.309-315.

  In the present invention, when cytotoxic T lymphocytes and target cells (for example, cancer cells, preferably lung cancer cells) are mixed and contacted at a cell number of 10: 1, cell lysis (%) is 15%. If% to 100%, preferably 30% to 100%, it is determined that the cytotoxic T lymphocytes tested exhibit cytotoxic activity against their target cells.

  The cytotoxicity test as described above can also be performed using a commercially available kit such as the CytoTox96 Non-Radioactive Cytotoxicity Assay kit (Promega) or the human Granzyme B ELISPOT kit (Euroclone).

  When measuring cytotoxic activity for cytotoxic T lymphocyte clones, in addition to cytotoxic activity against cancer cells, NK sensitive cells that do not have HLA class I molecules on their cell surface (eg, human leukemia cell line K562) or It is also preferable to simultaneously measure the cytotoxic activity against B cells (EBV-B cells) transformed with Epstein-Barr virus used as cells corresponding to normal cells. As a result, it has been confirmed that it exhibits cytotoxic activity against cancer cells, but cytotoxic activity against NK-sensitive cells that do not have HLA class I molecules on the cell surface (for example, the K562 strain) has not been confirmed. If cytotoxic activity against EBV-B cells used as cells corresponding to the above is not confirmed, the tested cytotoxic T lymphocyte clones can be determined to specifically recognize and damage cancer cells. Furthermore, it is preferable to similarly measure the cytotoxic activity against allogeneic cancer cells (non-self-derived cancer cells). As a result, if it is confirmed that the cells have cytotoxic activity against cancer cells and also have cytotoxic activity against allogeneic cancer cells, the cytotoxic T lymphocyte clones tested are autologous. It can be determined that the cancer cell is specifically recognized and damaged regardless of whether it is derived from another family or not.

  On the other hand, evaluation of HLA-A24 restriction on cancer cells with respect to cytotoxic T lymphocyte clones was carried out by using various cytokine production tests well known to those skilled in the art, and inhibition tests using various HLA antibodies. To do so. For example, cytotoxic T lymphocyte clones and cancer cells derived from HLA-A24 positive patients can be combined with anti-MHC class I antibody, anti-HLA-A24 antibody, anti-HLA-B / C antibody, anti-MHC class II antibody, etc. Mixed culture in a medium containing various anti-HLA antibodies, then measures the amount of cytokines such as interferon-γ (IFN-γ) or tumor necrosis factor (TNF) released into the culture supernatant, and puts the anti-HLA antibody in the medium HLA restriction is evaluated based on the degree to which the amount of cytokine is suppressed compared to the control group not added to the control group. When cytotoxic T lymphocyte clones were compared to cytokine levels in the control group, when anti-MHC class I antibody was added to the medium and cultured with cancer cells derived from HLA-A24 positive patients, the cytokine levels were , If it is decreased by at least 30% or more, preferably 50% to 100%, it can be judged that the cytokine-producing ability of the clone was inhibited by the anti-MHC class I antibody, and the cytotoxic T lymphocyte clone is MHC class I can be determined to be restrictive. Similarly, for cytotoxic T lymphocyte clones, when anti-HLA-A24 antibody was added to the medium and mixed culture with cancer cells derived from HLA-A24-positive patients, the amount of cytokine was compared to the control group. If it decreases by at least 30% or more, preferably 50% to 100%, it can be judged that the cytokine-producing ability of the clone was inhibited by the anti-HLA-A24 antibody, and the cytotoxic T lymphocyte clone is HLA-A24 It can be determined that it is restrictive.

  Furthermore, in the present invention, cytotoxic T lymphocyte clones can be examined for cytotoxic activity against cancer cells and HLA-A24 restriction using the tumor antigen peptide of the present invention. That is, by confirming that the tumor antigen peptide of the present invention is recognized in an HLA-A24-restricted manner with respect to the cytotoxic T lymphocyte clone obtained as described above, cancer cells containing autologous and other origins Cytotoxic T lymphocyte clones that specifically impair HLA-A24 restriction can be selected.

  The test for recognizing the tumor antigen peptide of the present invention in an HLA-A24-restricted manner may be performed as follows. First, a tumor antigen peptide is added to any HLA-A24 positive cells to prepare target cells. This target cell appears to express HLA-A24 molecule bound to the tumor antigen peptide of the present invention on the cell. Subsequently, the target cell and the cytotoxic T lymphocyte clone prepared as described above are mixed, and a cytotoxicity test is performed in the same procedure as described above. At this time, as a control group, it is also preferable to prepare a target cell using a peptide having the same length and different amino acid sequence as the tumor antigen peptide of the present invention, and conducting the same experiment. As a result, when the cytotoxic T lymphocyte clone exhibits a specific cytotoxic activity against the target cell presenting the tumor antigen peptide of the present invention, the cytotoxic T lymphocyte clone of the present invention It can be said that the tumor antigen peptide is specifically recognized by HLA-A24 restriction.

The cytotoxic T lymphocyte clone particularly preferably used in the present invention is the cytotoxic T lymphocyte clone F ₂ b / 5 obtained for the experiment described in Example 1 described later. Cytotoxic T lymphocyte clone F ₂ b / 5 is a cell derived from a regional lymph node lymphocyte of human (Homo sapiens), and is a tumor antigen peptide (amino acid sequence: NYGFQIHTK) is specifically recognized. Cytotoxic T lymphocyte clone F ₂ b / 5, dated March 31, 2004, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan) The deposit number is FERM P-19760.

4). Cellular medicine of the present invention The cytotoxic T lymphocyte obtained specifically as described above, which specifically damages HLA-A24-restricted cancer cells, is used in vitro to kill HLA-A24-positive cancer cells. Or it can be used in vivo.

  Therefore, the cytotoxic T lymphocytes that damage cancer cells in a restricted manner according to the present invention are administered to HLA-A24 positive humans for the purpose of treating cancer or preventing cancer recurrence, for example. It can be used particularly advantageously as a cell medicine.

  In the present invention, an antigen-presenting cell in which the tumor antigen peptide of the present invention is presented on an HLA-A24 molecule is also administered to an HLA-A24-positive human, for example, for the purpose of treating cancer or preventing cancer recurrence. By doing so, it can be particularly advantageously used as a cell medicine capable of inducing cytotoxic T lymphocytes that damage cancer cells in an HLA-A24-restricted manner in vivo.

  As used herein, “cellular medicine” refers to a medicine containing cytotoxic T lymphocytes or antigen-presenting cells in the state of living cells. This cell medicine may contain a pharmaceutically acceptable carrier or additive other than cytotoxic T lymphocytes. Examples of such carriers and additives include water, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, Examples include glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and artificial cell structures such as liposomes. The additive used is selected appropriately or in combination depending on the dosage form of the cell medicine.

  The cell medicine of the present invention may further contain other pharmacological components. As such pharmacological components, known drugs useful for immunostimulation can also be used.

  The cell medicine of the present invention is preferably administered parenterally, particularly preferably intravenous injection or direct injection into a lung tumor. As parenteral dosage forms, liquid preparations such as liquids, suspensions and syrups are preferred. Furthermore, the cell medicine may contain additives such as stabilizers, buffers, preservatives and the like that are generally used in pharmacy.

  The subject to which the cellular medicament of the present invention is administered is preferably an HLA-A24 positive human, particularly an HLA that has cancer, has previously had cancer, or has a predisposition to developing cancer. -A24 positive humans are preferred. The type of cancer is not particularly limited, but lung cancer is more preferable. The cancer to be treated or prevented by the pharmaceutical composition of the present invention is preferably a malignant tumor in which the expression of HLA-A24 antigen belonging to HLA class I is observed. Whether or not a human individual is HLA-A24 positive can be determined by an HLA typing method known to those skilled in the art, as described above.

The dosage of the cell medicine of the present invention varies depending on the age and weight of the administration subject, the administration route, and the number of administration, and can be appropriately changed by those skilled in the art. For example, it is preferable to administer a cell medicine containing 2 × 10 ⁶ to 2 × 10 ⁸ cells in terms of cytotoxic T lymphocytes per kg body weight per day at intervals of 1 to 2 weeks. The administration period may be determined according to the course of the patient's symptoms, but is preferably administered at least three times.

5. Cancer Vaccine and Pharmaceutical Composition of the Present Invention The tumor antigen peptide of the present invention can be effectively used for inducing cytotoxic T lymphocytes that damage HLA-A24-restricted cancer cells in vivo. Accordingly, a further aspect of the present invention is a peptide vaccine for the treatment of cancer containing the tumor antigen peptide of the present invention. The vaccine containing the tumor antigen peptide of the present invention, when administered to an HLA-A24 positive human individual, induces cytotoxic T lymphocytes that specifically damage cancer cells in an HLA-A24-restricted manner. As a result, it causes tumor shrinkage and, in some cases, complete cure of the cancer.

  The peptide vaccine containing the tumor antigen peptide of the present invention may contain a pharmaceutically acceptable carrier and / or additive other than the tumor antigen peptide of the present invention. This carrier and / or additive is the same as the above-mentioned cell medicine, but in addition, one or more kinds of adjuvants commonly used in vaccine preparations, such as Freund's complete adjuvant, Freund's incomplete adjuvant, BCG, etc. may be added. preferable. The peptide vaccine containing the tumor antigen peptide of the present invention preferably further contains IL-12, which is known to enhance the antitumor immune response in vivo.

  As a mode of administration of the peptide vaccine of the present invention, for example, it is preferable to administer a vaccine containing 0.001 to 1 g of a tumor antigen peptide per kg of body weight per time at intervals of 1 to 2 weeks. The administration period may be determined according to the course of the patient's symptoms, but it is preferably administered over at least 2 months.

  In the present invention, furthermore, an expression vector comprising DNA encoding the tumor antigen peptide of the present invention also expresses the tumor antigen peptide of the present invention in vivo, and thereby cells that damage cancer cells in an HLA-A24-restricted manner. It can be used effectively to induce impaired T lymphocytes. Accordingly, a further aspect of the present invention is a DNA vaccine for treating or preventing cancer, comprising an expression vector comprising DNA encoding the tumor antigen peptide of the present invention. As the expression vector used here, any expression vector used in gene therapy can be used. For example, an adenovirus vector or a retrovirus vector is often used. In a plasmid derived from E. coli, a strong promoter in eukaryotes (for example, an IE promoter / enhancer of cytomegalovirus) is upstream, and a poly A terminal sequence is used. Can also be used.

  A DNA vaccine containing an expression vector containing DNA encoding the tumor antigen peptide of the present invention can be inoculated by intramuscular injection, intradermal injection, transdermal inoculation, nasal inoculation, or the like. A vaccine containing an expression vector containing a DNA encoding the tumor antigen peptide of the present invention is administered into the living body of an HLA-A24-positive human individual, and then expresses the tumor antigen peptide of the present invention and is restricted to HLA-A24. Can induce cytotoxic T lymphocytes that specifically damage cancer cells, resulting in tumor shrinkage and, in some cases, complete cure of the cancer.

  The expression vector containing the tumor antigen peptide of the present invention or the DNA encoding the tumor antigen peptide can be used as a pharmaceutical product that enhances the therapeutic effect or preventive effect of cancer by being contained in a pharmaceutical composition (for example, an anticancer agent). it can. Accordingly, another aspect of the present invention is a pharmaceutical composition for enhancing the therapeutic effect of cancer, comprising an expression vector comprising the tumor antigen peptide of the present invention or a DNA encoding the tumor antigen peptide.

  In addition to the tumor antigen peptide of the present invention or the DNA encoding the tumor antigen peptide, a pharmaceutically acceptable carrier or additive may be added to the pharmaceutical composition as described above. Those pharmaceutically acceptable carriers or additives, dosage forms, administration subjects, administration modes and the like relating to the pharmaceutical composition of the present invention are the same as those described above for the cell medicine.

  Although the kind of cancer treated or prevented by the said pharmaceutical composition is not specifically limited, Lung cancer is more suitable. The cancer to be treated or prevented by the pharmaceutical composition of the present invention is preferably a malignant tumor in which the expression of HLA-A24 antigen belonging to HLA class I is observed.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Induction of cytotoxic T lymphocyte clones specific to cancer cells
1. Establishment of a cell line A 46-year-old male lung large cell carcinoma patient (A904 case, pT2N0M0 Stage IB) was established from a surgical resection sample of a lung cancer cell line (hereinafter, this established cell line is referred to as A904L). The HLA class I genotype of this cell line A904L was HLA-A * 2402, B * 0702, Cw * 0702.

  Furthermore, peripheral blood mononuclear cells collected from these A904 patients were transformed into Epstein-Barr virus (EBV-B) using the culture supernatant of B95.8, an EBV (Epstein-Barr virus) production strain. An A904 B cell line that was transformed by infection and immortalized as a result was produced (hereinafter, this cell line is referred to as A904-EBV-B cell). The HLA class I genotypes of the A904-EBV-B cells were as follows: HLA-A * 2402, 2603, B * 0702, 3901, Cw * 0702, 0702.

These two cell lines were maintained in the following RPMI 1640 medium supplemented with 5% or 10% FCS.
Medium supplemented with 5% or 10% FCS: RPMI 1640 (GIBCO-BRL, Grand Island, NY), 5% or 10% heat-inactivated fetal calf serum (EQUITECH-BIO, INC. INGRAM, TX), 10 mM HEPES, Add 100 units / ml penicillin G and 100 mg / ml streptomycin sulfate. This 5% FCS-added RPMI 1640 medium or 10% FCS-added RPMI 1640 medium was also used in the following examples.

2. Transfection of CD80 cDNA into lung cancer cell line In order to increase the induction efficiency of cytotoxic T lymphocytes, a plasmid containing human CD80 cDNA encoding CD80, a costimulatory molecule, was added to lipofectamine to the cell line A904L prepared above. Transfection was performed by a lipofection method using a reagent (GIBCO-BRL). Detailed procedures were as described in Takenoyama, M. et al., (2001) Jpn. J. Cancer Res., 92, p. 309-315. The base sequence of this CD80 cDNA is registered in the DDBJ / EMBL / GenBank international base sequence database as accession number NP005182.

  Transfected A904L cells were selected by G418 and named A904L-B7 strain. The A904L-B7 strain was inactivated by irradiation with 100 Gy.

3. Isolation of T lymphocytes Regional lymph nodes were collected at the same operation from A904 patients described in “1. Establishment of cell lines” above. From this regional lymph node, regional lymph node lymphocytes were prepared according to the technique described in Takenoyama, M. et al., (1996) Immunobiology, 195, p.140-151. Regional lymph node lymphocytes were stored frozen in a deep freezer at -130 ° C until use.

4). Induction of cytotoxic T lymphocytes Regional lymph node lymphocytes (2.6 × 10 ⁷ cells) were obtained by rapid thawing from cryopreserved cells. The regional lymph node lymphocytes and the A904L-B7 prepared above (CD80 transfected A904L cells after 100 Gy irradiation) were 25 units / ml so that the lymphocyte: cancer cell ratio was 10: 1. Wells in 24-well plates containing 10% FCS supplemented RPMI 1640 medium supplemented with rIL-2 (donated by Takeda Pharmaceutical), 5 ng / ml IL-4 (Serotec) and 5 ng / ml IL-7 (Genzyme Techne) In addition, regional lymph node lymphocytes were stimulated by overnight culture. This regional lymph node lymphocyte was stimulated once a week for a total of 4 times at 37 ° C. under 5% CO ₂ . Cytotoxic T lymphocytes were induced by such stimulated culture. The details of the operation related to the stimulation of the above-mentioned regional lymph node lymphocytes were in accordance with the procedure described in Lehmann et al., (1995) Eur. J. Immunol., 25, p.340-347.

Subsequently, cytotoxic T lymphocytes were obtained as T lymphocyte clones by limiting dilution using the cell population 7 days after the last stimulation (hereinafter, this T lymphocyte clone was referred to as F ₂ b / 5). The details of the operation relating to the acquisition of T lymphocyte clones by this limiting dilution method followed the procedure described in So et al., (2001) Jpn. J. Clin. Oncol., 31, p.311-317.

This cytotoxic T lymphocyte clone F ₂ b / 5 was founded on March 31, 2004, at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan). No. 6) was deposited under the accession number FERM P-19760.

[Example 2] Confirmation of cancer-specific cytotoxic activity and HLA-A24 restriction of cytotoxic T lymphocyte clones
1. Cytotoxic activity against cancer cells Cytotoxic T lymphocyte clone F ₂ b / 5 obtained as described above was evaluated for its cytotoxic activity by a standard 4 hr ⁵¹ Cr release assay. As a test group, autologous lung cancer cell line A904L from which cytotoxic T lymphocyte clones were derived was used. As a control group, a cell (A904-EBV-B cell) transformed with Epstein-Barr virus B cell of a lung cancer patient A904 derived from T lymphocyte clone F ₂ b / 5, and human leukemia cell line K562 used. The specific procedure of ⁵¹ Cr release assay was performed as described in So et al., (2001) Jpn. J. Clin. Oncol., 31, p.311-317.

The result is shown in FIG. As shown in FIG. 1, the cytotoxic T lymphocyte clone F ₂ b / 5 exhibited effective cytotoxic activity against the lung cancer cell line A904L, which is an autologous tumor. On the other hand, A904-EBV-B cells used as target cells corresponding to autologous normal cells and HLA class I molecule negative human leukemia cell line K562 did not show cytotoxic activity. From this result, the cytotoxic T lymphocyte clone F ₂ b / 5 obtained in Example 1 shows cytotoxic activity against autologous lung cancer cells, but against autologous normal cells and HLA. It was confirmed that it did not show cytotoxic activity against class I molecule negative tumor cells.

2. HLA-restricted Furthermore, the cytotoxic T lymphocyte clones F ₂ b / 5, used to verify the cytotoxic activity and the HLA-restricted represented by cytokine productivity, various anti-HLA antibodies in cytokine production test inhibition A test was conducted. Lung cancer cell line A904L was used as a target cell to be damaged by cytotoxic T lymphocyte clone F ₂ b / 5. Anti-HLA antibodies for inhibition include anti-MHC class I antibodies (W6 / 32: anti-HLA-A, B, Cw), anti-HLA-A24 antibodies (C7709.A.2.6), anti-HLA-B / C antibody (B.1.23.2) and anti-MHC class II antibody (IVA12: HB145; manufactured by ATCC) were used. The specific experimental procedure was performed as described in So et al., (2001) Jpn. J. Clin. Oncol., 31, p.311-317.

The result is shown in FIG. As shown in FIG. 2, cytotoxic T lymphocyte clone F ₂ b / 5 produced an average of 64.6 pg / ml of TNF when cultured in the absence of anti-HLA antibody with autologous lung cancer cell line A904L. (Control group). In contrast, with A904L and F ₂ b / 5, when mixed cultured in the presence of anti-MHC class I antibody, TNF amount of average 1.2 pg / ml were measured. The amount of TNF was reduced by 98% compared to the control group, indicating that recognition of A904L by F ₂ b / 5 was inhibited by anti-MHC class I antibodies. Similarly, when F ₂ b / 5 was cocultured with A904L in the presence of anti-HLA-A24 antibody, an average TNF amount of 7.7 pg / ml was measured, which was 88% compared to the control group. From this, it was shown that the recognition of A904L by F ₂ b / 5 was inhibited by the anti-HLA-A24 antibody. In addition, when F ₂ b / 5 was mixed with A904L in the presence of anti-HLA-B & C antibody or anti-MHC class II antibody, the average amount of TNF was 54.2 pg / ml and the average TNF was 55.7 pg / ml. The amount was measured, which was a 16%, 14% decrease compared to the control group. That is, recognition of A904L by F ₂ b / 5 was hardly inhibited by anti-HLA-B & C antibody and anti-HLA class II antibody. From these results, it was proved that recognition of A904L by F ₂ b / 5 is HLA-A24-restricted.

3. Cytotoxic activity against allogeneic cancer cells Next, cytotoxicity against the B203L strain, which is an allogeneic lung cancer cell line, was evaluated for the cytotoxic T lymphocyte clone F ₂ b / 5 by a ⁵¹ Cr release assay. In addition, as a control group, cytotoxic activity against cells (B203-EBV-B cells) obtained by transforming B cells derived from the same patient as the B203L strain (B203 case) with Epstein-Barr virus was also evaluated. The specific experimental procedure was performed according to the description of So et al., (2001) Jpn. J. Clin. Oncol., 31, p.311-317. ^{In the 51} Cr release assay, B203L strain B203-EBV-B cells were used instead of the aforementioned K562 strain and A904-EBV-B cells.

The result is shown in FIG. In FIG. 3, the cell lysis (%) of the B203L strain is represented by a white square (□), and the cell lysis (%) of the B203-EBV-B cell is represented by a black square (■). As can be seen from FIG. 3, the cytotoxic T lymphocyte clone F ₂ b / 5 showed cytotoxic activity against the B203L strain, but showed cytotoxic activity against the B203-EBV-B cells. There wasn't. This result shows that cytotoxic T lymphocyte clone F ₂ b / 5 does not show cytotoxic activity against normal cells derived from other families, but has cytotoxic activity against lung cancer cells derived from other families. Proved to show. This is because the cytotoxic T lymphocyte clone F ₂ b / 5 recognizes a common antigen shared by lung cancer cells regardless of whether it is autologous or non-autologous. This shows that normal cells are not damaged, but normal cells are not damaged.

[Example 3] Isolation of cDNA clone encoding tumor antigen First, total RNA was extracted from A904L cells (HLA-A * 2402, B * 0702, Cw * 0702) using Rneasy Mini Kit (Qiagen), CDNA was generated by PT-PCR using oligo (dT) primers.

Next, using this cDNA, a plasmid pcDNA3 into which a gene amplified fragment of HLA-A * 2402, which is a subtype of HLA-A24, was inserted was prepared in advance. PCR amplification was performed using the cDNA as a template and a primer pair specific to the HLA-A24 gene, and the resulting PCR product was cloned into pcDNA. The HLA-A24 gene-specific primer pair used here is as follows.
Forward primer: 5'-ACTGGGCGGATCCGGACTCAGAATCTCCCCAGACGCCGAG-3 '(SEQ ID NO: 3)
Reverse primer: 5'-ACTGCCCGAATTCTCTCAGTCCCTCACAAGGCAGCTGTC-3 '(SEQ ID NO: 4)

  On the other hand, in order to prepare a cDNA library, the above cDNA was ligated into a HindIII-EcoRI adapter and then restriction digested with NotI. The obtained cDNA fragment was inserted into the HindIII-NotI site of the expression vector pCEP4 and ligated. Subsequently, the recombinant plasmid thus prepared was transformed and introduced into Escherichia coli TOP10 by electroporation, and further selected on a medium containing ampicillin (50 μg / ml). For the obtained cDNA clone, a cDNA library of 1,118 pools was prepared using 100 colonies as one pool. Furthermore, plasmid DNA was extracted from each pool.

Subsequently, 293-EBNA cells were seeded in a 96-well flat bottom microwell plate at 3.5 × 10 ⁴ cells / well and cultured. 24 hours after sowing, 1.3 μl of Lipofectamine reagent (Life Tecnology, Inc.), 100 ng of plasmid DNA obtained from the previously extracted A904L-derived cDNA library, and HLA-A * 2402 gene amplified fragment were obtained. 50 ng of the inserted plasmid pcDNA3 (Invitrogen) was added, and 293-EBNA cells were co-transfected. By this cotransfection, the 293-EBNA cells bind to the HLA-A2402 molecule and present the antigen fragment expressed from the cDNA derived from A904L cells on the cells. 24 hours after co-transfection, the cytotoxic T lymphocyte clone F ₂ b / 5 obtained in Example 1 was added at 3000 cells / well and further cultured, and F ₂ b / 5 was added. After 24 hours, the culture supernatant was collected. The culture supernatant was subjected to a bioassay using WEHI-164cl3 cells, and the amount of TNF produced from F ₂ b / 5 was measured. From the 293-EBNA cell clone of the sample that produced a large amount of TNF from F ₂ b / 5, the plasmid DNA introduced into the cell was extracted, and the cDNA clone (cDNA 214.2) inserted into the plasmid DNA was simply isolated. Released. As a result of nucleotide sequencing and sequence analysis, this cDNA clone was composed of 3339 bp, and an open reading frame (ORF) encoding a 617 amino acid sequence was found therein. This cDNA clone was presumed to be a splicing variant of the known gene Tara, and the protein consisting of this 617 amino acid sequence was considered to be a tumor antigen (FIG. 4A).

Furthermore, the plasmid pcDNA3 with the HLA-A * 2402 gene inserted was transfected into 293-EBNA cells alone, and the amount of TNF produced from F ₂ b / 5 cultured with the 293-EBNA cells was measured. . As a result, F ₂ b / 5 did not produce TNF. Similarly, when 293-EBNA cells were co-transfected with HLA-type HLA-A2 gene different from HLA-A24 and cDNA clone 214.2 encoding tumor antigen, F ₂ b / cultured with the 293-EBNA cells. 5 still did not produce TNF. Therefore, cytotoxic T lymphocyte clone F ₂ b / 5 was co-transfected with plasmid pcDNA3 (HLA-A24 gene) inserted with HLA-A * 2402 gene and cDNA clone 214.2 (tumor antigen). Only when it was shown to produce TNF. Therefore, it was proved that this cDNA clone 214.2 encodes a tumor antigen peptide presented in HLA-A24 and recognized by F ₂ b / 5 (FIG. 4B).

[Example 4] Identification of antigenic peptide By setting various primers in the open reading frame of the cDNA clone isolated in Example 3 and performing PCR amplification using the primers, a minigene with a short C-terminal is prepared. did. The used primers are as follows.
Forward primer OKY 167: 5'-AGGATGAGCAACTTCTTTATC-3 '(SEQ ID NO: 5)
Reverse primer OKY 191: 5'-GGTGTGGATCTGGAAGC-3 '(SEQ ID NO: 6)
Reverse primer OKY 192: 5'-GTGGATCTGGAAGCCATA-3 '(SEQ ID NO: 7)
Reverse primer OKY 193: 5'-GATCTGGAAGCCATAGTTG-3 '(SEQ ID NO: 8)
Reverse primer OKY 194: 5'-CTGGAAGCCATAGTTGCG-3 '(SEQ ID NO: 9)

PCR products amplified using OKY 167 and OKY 191 are 398 bp long, PCR products amplified using OKY 167 and OKY 192 are 395 bp long, PCR products amplified using OKY 167 and OKY 193 are 392 bp long The PCR product amplified using OKY 167 and OKY 194 was 389 bp in length. Next, these PCR products were co-transfected into 293-EBNA cells together with the HLA-A * 2402 gene in the same manner as in Example 3, and the 293-EBNA cells and F ₂ b / 5 were mixed and cultured. The amount of TNF produced in the culture supernatant was measured. As a result, TNF production was observed when PCR products of 398 bp and 395 bp length were used, respectively, but TNF production was not observed when PCR products of 392 bp length and 389 bp length were used respectively (Fig. 5). From this, the tumor antigen epitope recognized by the cytotoxic T lymphocyte clone F ₂ b / 5 is present in the protein portion corresponding to the 3 ′ end portion of the PCR product having a length of 395 bp, that is, in the black portion in FIG. It was shown that.

Therefore, ten types of 9 amino acid peptides were designed from the deduced amino acid sequence based on the nucleotide sequence of base numbers 432 to 479 of cDNA clone 214.2 corresponding to the 3 ′ end portion of the 395 bp long PCR product (FIG. 6). These peptides were synthesized by automated peptide synthesis. Subsequently, each of these peptides was added to C1R-A24, which is an HLA-A24-expressing cell, and cultured to display each peptide on the HLA-A24 molecule on the cell. The cells presenting each peptide and the cytotoxic T lymphocyte clone F ₂ b / 5 were mixed and cultured, and then the cytotoxic activity of F ₂ b / 5 against each peptide was evaluated. The result is shown in FIG. Cytotoxic T lymphocyte clone F ₂ b / 5 exhibits high cytotoxic activity against cells presenting peptide 3 (NYGFQIHTK), whereas it exhibits no cytotoxic activity against cells presented with other peptides. Since it was not shown, it was proved that this peptide 3 is a tumor antigen peptide specifically recognized by the cytotoxic T lymphocyte clone F ₂ b / 5.

  The cancer-specific tumor antigen peptide of the present invention can be used to specifically induce the cytotoxic activity of cytotoxic T lymphocytes that damage cancer cells in a HLA-A24-restricted manner. It can be used to select cytotoxic T lymphocytes that restrictively damage cancer cells. The tumor antigen peptide of the present invention can also be used as a cancer vaccine for inducing anti-tumor immunity in HLA-A24 positive cancer patients.

  The cytotoxic T lymphocyte that recognizes the tumor antigen peptide of the present invention in an HLA-A24-restricted manner can specifically damage cancer cells and does not damage normal cells, so it can be used for cancer immunotherapy. It can be used as a highly effective cell medicine with few side effects.

  Furthermore, the DNA encoding the cancer-specific antigen peptide of the present invention can be used as a template for producing the tumor antigen peptide of the present invention by a recombinant method. Furthermore, an expression vector containing the DNA is used as a DNA vaccine. Can also be used.

FIG. 1 shows the cytotoxicity of cytotoxic T lymphocyte clones against the autologous lung cancer cell lines A904L, A904-EBV-B cells and human leukemia cell line K562 used as target cells, as measured by cytolysis (%). It is a figure which shows the result. The vertical axis represents the cell lysis (%) of the target cells, and the horizontal axis represents the cytotoxic T lymphocyte: target cell mixing ratio expressed by the number of cytotoxic T lymphocytes / number of target cells. FIG. 2 is a graph showing the inhibitory effect of anti-HLA antibodies on the cytotoxic activity of cytotoxic T lymphocyte clones. CTL represents a cytotoxic T lymphocyte clone, AT represents an autologous lung cancer cell line A904L, and mAb represents a monoclonal antibody. FIG. 3 is a graph showing the cytotoxic activity of cytotoxic T lymphocyte clones against the B203L strain, an allogeneic lung cancer cell line. The vertical axis represents the cell lysis (%) of the target cells, and the horizontal axis represents the cytotoxic T lymphocyte: target cell mixing ratio expressed by the number of cytotoxic T lymphocytes / number of target cells. □ represents B203L, and ■ represents B203-EBV-B cells. FIG. 4A is a schematic diagram of cDNA 214.2 encoding a tumor antigen. FIG. 4B shows that cytotoxic T lymphocyte clones exhibited cytotoxic activity only on cells co-transfected with cDNA encoding tumor antigen and HLA-A24 cDNA. FIG. 5 is a schematic diagram of tumor antigen fragments used to identify tumor antigen peptides that induce cytotoxic activity of cytotoxic T lymphocyte clones. Cytotoxic activity (+) indicates that cytotoxic activity has been demonstrated for the target cells into which the tumor antigen fragment has been introduced, and cytotoxic activity (−) indicates that the cells have no activity against the target cells into which the tumor antigen fragment has been introduced This indicates that no disorder activity was shown. FIG. 6 is a schematic diagram showing a design sequence of a synthetic peptide used for identification of a tumor antigen peptide. FIG. 7 shows the results of inducing cytotoxic activity of cytotoxic T lymphocyte clones using synthetic peptides. ● represents peptide 3 corresponding to the tumor antigen peptide of the present invention.

  The sequence | arrangement of sequence number 3-9 shows a primer.

Claims (16)

  A peptide comprising the amino acid sequence of NYGFQIHTK (SEQ ID NO: 1).   DNA encoding the peptide according to claim 1. Expression vectors containing claim 2 Symbol placement of DNA.   A cytotoxic T lymphocyte that recognizes a peptide comprising the amino acid sequence of NYGFQIHTK (SEQ ID NO: 1) in an HLA-A24-restricted manner.   Cytotoxic T lymphocyte shown by accession number FERM P-19760. NYGFQIHTK comprises the amino acid sequence of (SEQ ID NO: 1), and a peptide consisting of one or several amino acids are pressurized with on its amino acid sequence, cytotoxic T lymphocytes according to claim 4 or 5 The peptide, which is recognized as HLA-A24 restricted by a sphere. The peptide according to claim 6, wherein the added amino acid is 1 or 2 amino acids. A vaccine for treating or preventing cancer comprising the peptide according to claim 1 or 6 . A vaccine for treating or preventing cancer comprising the expression vector according to claim 3 .   The vaccine according to claim 8 or 9, wherein the cancer is lung cancer.   The vaccine according to any one of claims 8 to 10, for an HLA-A24 positive patient. An antigen-presenting cell in which the peptide according to claim 1 or 6 is presented on an HLA-A24 molecule. A cell medicine containing the cytotoxic T lymphocyte according to claim 4 or 5 .   A cell medicine comprising the antigen-presenting cell according to claim 12. A method for obtaining cytotoxic T lymphocytes that damage HLA-A24-restricted cancer cells, comprising the following steps (1) to (3):
(1) mixing culturing the peptide and HLA-A24-positive cells according to claim 1 or 6, for preparing a HLA-A24-positive cells presenting the peptide according to HLA-A24 molecule in claim 1 or 6 Process,
(2) A step of inducing cytotoxic T lymphocytes by co-culturing a cell population containing HLA-A24-positive cancer cells and a cell population containing HLA-A24-positive CTL precursor cells, and
(3) Cells obtained in step (1) of cytotoxic T lymphocytes by co-culturing the cells prepared in step (1) and the cytotoxic T lymphocytes induced in step (2) Measuring the cytotoxic activity against. A method for inducing cytotoxic T lymphocytes that damage HLA-A24-restricted cancer cells, comprising the following steps (1) and (2):
(1) and peptide according to claim 1 or 6, and HLA-A24-positive cells having antigen-presenting ability by mixed culture, antigen-presenting cells presenting the peptide according to HLA-A24 molecule in claim 1 or 6 And the step of preparing
(2) A step of mixing and culturing the antigen-presenting cells prepared in step (1) and a cell population containing HLA-A24-positive CTL precursor cells.

Images