Specification
Title of invention: immune inducing agent
Technical field
[0001]
　The present invention relates to a novel immunity inducing agent useful as an active ingredient of a therapeutic or prophylactic agent for cancer.
BACKGROUND ART
[0002]
　PDS 5 A (PDS 5, regulator of cohesion maintenance, homolog A) protein, also known as SSC-112, is a protein identified as a cell cycle regulator involved in chromosome partitioning.
[0003]
　PDS5A protein has been suggested to be associated with carcinogenesis. For example, Patent Document 1 discloses that PDS5A protein has higher expression in nasopharyngeal cancer, kidney cancer, liver cancer, breast cancer cell as compared with normal tissue. In addition, it is possible to suppress the proliferation of cancer cells by suppressing the expression of PDS5A protein in cancer cells using antisense nucleic acid, ribozyme, siRNA, or anti-PDS5A protein antibody against PDS5A gene, and that the full-length protein of PDS5A or the protein It has also been disclosed that apoptosis can be induced in cancer cells by administering partial peptides of the present invention.
[0004]
　In Patent Document 2, the PDS5A protein binding to HLA-A0201 which is a subtype of MHC class I and the partial peptide of the protein have an immunity-inducing activity against cancer cells and are therefore useful for treatment and prevention of cancer It is disclosed that there is something. However, in Patent Document 2, not all peptides binding to HLA-A0201 are disclosed, and information on peptides that bind to subtypes other than HLA-A0201 is not disclosed.
Prior Art Document
Patent literature
[0005]
Patent Document 1: WO 2002/081641
Patent Document 2 : WO 2010/027807
Summary of the invention
Problem to be Solved by Invention
[0006]
　An object of the present invention is to find a novel polypeptide useful as an active ingredient of a therapeutic or prophylactic agent for cancer and to provide use of the polypeptide as an immunity inducing agent.
[0007]
　Also, an object of the present invention is to provide an isolated antigen-presenting cell comprising a complex of said polypeptide and HLA molecule and an isolated T cell that selectively binds to said complex of said polypeptide and HLA molecule, To provide a therapeutic or prophylactic agent.
Means for solving the problem
[0008]
　As a result of intensive research, the inventors of the present invention have found that human PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is a human PDS5A protein having leukemia, malignant lymphoma, breast cancer, liver cancer, prostate cancer, pancreatic cancer, ovarian cancer, kidney cancer, Gastric cancer, malignant brain tumor, esophageal cancer and lung cancer tissues or cells. Further, it is preferable that the partial peptide present in a specific region of the PDS5A protein has the ability (antigen-inducing activity) to be presented by antigen presenting cells and to activate and proliferate T cells specific to the polypeptide, and And found that the immunity inducing activity is useful for treating or preventing cancer. Based on these results, it can be said that the polypeptide can be an active ingredient of an immunity-inducing agent for the treatment and / or prevention of cancer, and that the antigen presenting cells contacted with the peptide or T And found that the cells are also useful for treating or preventing cancer, thus completing the present invention.
[0009]
　That is, the present invention has the following features (1) to (14).
(1) An immunity inducing agent comprising the following (i) or (ii) as an active ingredient.
[0010]
　(I) at least one polypeptide selected from the group of polypeptides according to (a) or (b) below and having immunity inducing activity,
　　(a) at least one polypeptide selected from the group consisting of 24 to 97 , Positions 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, 533 to 567, 613 to 643, 671 to 735, 737 to 780
　　(B) consisting of consecutive 7 or more amino acids within the region from positions 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, and 1074 to 1215, A polypeptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence of any one of the polypeptides described in (a) above, or
　(ii) A recombinant vector capable of expressing the polypeptide in vivo comprising at least one polynucleotide encoding a polypeptide of the present
invention (2) the polypeptide according to (i) binds to MHC class I molecule (1 ).
(3) The immunity inducing agent according to (2), wherein the polypeptide according to (i) is any one polypeptide selected from the group of polypeptides described in (c) to (e) below .
[0011]
　　(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 3 to 34
　　(d) a polypeptide in which one to several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide described in (c)
　　(e) a polypeptide comprising the polypeptide according to (c) or (d) as a partial sequence
(4) The polypeptide according to (1), wherein the polypeptide according to (i) binds to MHC class II molecule An immunity inducing agent.
(5) The immunity inducing agent according to (4), wherein the polypeptide according to (i) is any one polypeptide selected from the group of polypeptides described in (f) to (h) below .
[0012]
　　(f) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 35 to 67
　　(g) a polypeptide obtained by deleting, substituting or adding one to several amino acids in the amino acid sequence of the polypeptide according to (f)
　　(h) a polypeptide comprising the polypeptide according to (f) or (g) as a partial sequence
(6) described in any one of (1) to (5), which is used as an active ingredient of a therapeutic or prophylactic agent for cancer .
(7) The immunity inducing agent according to (6), wherein the cancer is a cancer expressing PDS5A protein.
(6) or (7), wherein the cancer is leukemia, malignant lymphoma, prostate cancer, liver cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer, colon cancer, gastric cancer, malignant brain tumor, lung cancer or esophageal cancer; .
(9) The immunity inducing agent according to any one of (1) to (8), further comprising an immunity enhancing agent.
(10) A polypeptide selected from the following polypeptide group (a) or (b) and having immunity inducing activity.
[0013]
　　(a) an amino acid sequence represented by SEQ ID NO: 2 at positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, In the region of positions 567, 613 to 643, 671 to 735, 737 to 780, 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, and 1074 to 1215 A polypeptide having an immunity-inducing activity consisting of 7 or more consecutive
　　amino acids in the amino acid sequence of SEQ ID NO: 1 ; (b) a polypeptide having deletion, substitution or addition of one or several amino acids in the amino acid sequence of the polypeptide described in (a) Polypeptide.
(11) The polypeptide according to (10), which is any one polypeptide selected from the group of polypeptides described in (c) to (e) below.
[0014]
　　(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 3 to 34
　　(d) a polypeptide in which one to several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide described in (c)
　　(e) a polypeptide comprising the polypeptide according to (c) or (d) as a partial sequence
(12) Any one of the following polypeptides selected from the group of polypeptides (f) to (h) The polypeptide according to (10), which is a peptide.
[0015]
　　(f) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 35 to 67
　　(g) a polypeptide obtained by deleting, substituting or adding one to several amino acids in the amino acid sequence of the polypeptide according to (f)
　　(h) a polypeptide comprising the polypeptide according to (f) or (g) as a partial sequence
(13) a polypeptide having an immunity-inducing activity according to any one of (10) to (12) Isolated antigen presenting cells comprising a complex.
(14) An isolated T cell that selectively binds a complex of a polypeptide having an immunity-inducing activity and an MHC molecule according to any one of (10) to (12).
[0016]
　This specification encompasses the disclosure of Japanese Patent Application No. 2015-158539 on which the priority of the present application is based.
Effect of the invention
[0017]
　According to the present invention, a novel immunity inducing agent useful as an active ingredient of a therapeutic or prophylactic agent for cancer is provided.
[0018]
　In addition, as specifically shown in Examples described later, immune cells which kill cancer cells can be induced by the polypeptide used in the present invention, and cancers that have already occurred can be reduced or regressed . Furthermore, the peptide used in the present invention can enhance the induction of immune cells that kill cancer cells, and can reduce or regress existing cancers. Therefore, the polypeptide of the present invention is useful as an active ingredient of a therapeutic or prophylactic agent for cancer.
Brief Description of the Drawings
[0019]
FIG. 1 is a diagram showing an expression pattern of a PDS5A gene in a human tumor tissue or cancer cell line. Reference number 1: shows the expression pattern of the human PDS5A gene. Reference number 2 shows the expression pattern of the GAPDH gene which is a human housekeeping gene.
[FIG. 2] CD8 positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 19 recognize a complex of the polypeptide and HLA-A0201 to produce IFN-γ FIG. In the figure, lanes 13 to 29 on the abscissa represent IFN-γ production of HLA-A0201-positive CD8-positive T cells by stimulation of dendritic cells pulsed with polypeptides represented by the amino acid sequences of SEQ ID NOs: 3 to 19 Ability is indicated. Lane 1 shows the result (Mock) for the case where the above treatment was carried out without adding the polypeptide, and lane 2 shows the result of adding the negative control polypeptide outside the range of the present invention shown in SEQ ID NO: 74, Lane 3 shows the results obtained by adding the full-length PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and the lanes 4 to 12 show the results shown in SEQ ID NOs: 75 to 83 The results obtained by adding the polypeptide outside the scope of the invention and carrying out the above treatment are shown.
[Figure 3] CD8 positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 20 to 34 recognize a complex between the polypeptide and HLA-A24 to produce IFN-γ FIG. In the figure, lanes 4 to 18 on the abscissa represent IFN-γ production of HLA-A24 positive CD8 positive T cells by stimulation of dendritic cells pulsed with polypeptides represented by the amino acid sequences of SEQ ID NOs: 20 to 34 Ability is indicated. Lane 1 shows the result (Mock) for the case where the above treatment was carried out without adding the polypeptide, and lane 2 shows the result with the negative control peptide outside the range of the present invention shown in SEQ ID NO: 84 added And lane 3 shows the results of the above treatment by adding the full-length PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
FIG. 4A is a diagram showing the disorder activity of CD8 positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 19 against cancer cells. In the figure, lanes 13 to 29 on the abscissa indicate cytotoxic activity against U251 cells of HLA-A0201 positive CD8 positive T cells induced by using polypeptides represented by the amino acid sequences of SEQ ID NOs: 3 to 19, respectively . Lane 1 shows the cytotoxic activity of CD8 positive T cells (Mock) induced without adding the polypeptide, and lane 2 shows the CD8 positive T cell cells induced using the negative control polypeptide (SEQ ID NO: 74) Lane 3 shows the cytotoxic activity of CD8 positive T cells induced with the full-length PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and lanes 4 to 12 show the cytotoxic activity of SEQ ID NO: 75 The cytotoxic activity of CD8-positive T cells induced using polypeptides out of the scope of the present invention shown in.
FIG. 4B is a diagram showing the disorder activity of CD8 positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOS: 3 to 19 against cancer cells. In the figure, lanes 12 to 28 on the abscissa indicate the cytotoxic activity of HLA-A0201 positive CD8 positive T cells induced by using the polypeptides represented by the amino acid sequences of SEQ ID NOs: 3 to 19 on Jurkat cells . Lane 1 shows the cytotoxic activity of CD8 positive T cells (Mock) induced without adding the polypeptide, and lane 2 shows the CD8 positive T cell cells induced using the negative control polypeptide (SEQ ID NO: 74) Lane 3 shows the cytotoxic activity of CD8 positive T cells induced using the full-length PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and lanes 4 to 11 show the cytotoxic activity of SEQ ID NO: 75 to SEQ ID NO: The cytotoxic activity of CD8 positive T cells induced using the polypeptide outside the range of the present invention shown in 83 is shown.
FIG. 5A is a diagram showing the disorder activity of CD8 positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 20 to 34 against cancer cells. Lanes 4 to 18 on the abscissa indicate cytotoxic activity against THP1 cells of HLA-A24 positive CD8 positive T cells stimulated with polypeptides represented by the amino acid sequences of SEQ ID NOs: 20 to 34, respectively. Lane 1 shows the cytotoxic activity of CD8 positive T cells (Mock) induced without adding the polypeptide, and lane 2 shows the CD8 positive T cell cells induced using the negative control polypeptide (SEQ ID NO: 84) And lane 3 shows cytotoxic activity of CD8 positive T cells induced using the PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
FIG. 5B is a diagram showing the disorder activity of CD8 positive T cells specific to each peptide consisting of the amino acid sequences shown in SEQ ID NOS: 20 to 34 against cancer cells. Lanes 4 to 18 on the abscissa indicate the cytotoxic activity against SW480 cells of HLA-A24 positive CD8 positive T cells stimulated with the polypeptides represented by the amino acid sequences of SEQ ID NOs: 20 to 34, respectively. Lane 1 shows the cytotoxic activity of CD8 positive T cells (Mock) induced without adding the polypeptide, and lane 2 shows the CD8 positive T cell cells induced using the negative control polypeptide (SEQ ID NO: 84) And lane 3 shows cytotoxic activity of CD8 positive T cells induced using the PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
[FIG. 6] CD4 positive T cells specific for each polypeptide consisting of the amino acid sequences shown in SEQ ID NOS: 35 to 67 recognize a complex of the polypeptide and HLA-DRB 1 * 04 to produce IFN-γ FIG. Lanes 4 to 36 show the IFN-γ productivity of HLA-DRB 1 * 04 positive CD 4 positive T cells stimulated by dendritic cells pulsed with a polypeptide represented by the amino acid sequence of SEQ ID NO: 35 to 67, respectively. Lane 1 shows the results of Mock for the case where the above treatment was carried out without adding a polypeptide, and lane 2 shows the results of the above treatment by adding a negative control polypeptide outside the scope of the present invention shown in SEQ ID NO: 85 And lane 3 shows the results of the above treatment by adding the full-length PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
MODE FOR CARRYING OUT THE INVENTION
[0020]
　 In the
　present invention, the term "polypeptide" refers to a molecule formed by a plurality of amino acid peptide bonds. Not only a polypeptide molecule having a large number of amino acids to constitute but also a low molecular weight molecule (oligopeptide) having a small number of amino acids is also included in the polypeptide of the present invention.
[0021]
　The polypeptide constituting the immunity-inducing agent of the present invention includes at least one polypeptide selected from the group of polypeptides described in (a) or (b) below and having immunity-inducing activity.
(a) a human PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, which has a position of 24 to 97 (74 amino acids), 113 to 132 (20 amino acids), 134 to 197 ( 64 amino acids), 204 to 225 (22 amino acids), 265 to 332 (68 amino acids), 378 to 463 (86 amino acids), 472 to 498 (27 amino acids), 533 to 567 (35 amino acids), 613 (24 amino acids), 792 to 830 (39 amino acids), 832 to 899 (68 amino acids), 920 to 943 (24 amino acids), positions 671 to 735 (65 amino acids), 737 to 780 Amino acids), 946 to 993 (58 amino acids), 1029 to 1069 (41 amino acids), and 1074 to 1 A polypeptide consisting of 7 or more consecutive amino acids within a region of 215 position (142 amino acids)
(b) one to several amino acids in the amino acid sequence of the polypeptide described in (a) above are deleted, substituted or substituted An added polypeptide.
[0022]
　In the present invention, "consisting of an amino acid sequence" means that amino acid residues are arranged in such order. Therefore, for example, "a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2" refers to a polypeptide having the amino acid sequence of Met Asp Phe Thr shown in SEQ ID NO: 2 ... (...) --- Asp Leu Gln Arg Means a polypeptide of size 1337 amino acid residues. Also, in the present specification, for example, "a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2" is often abbreviated as "a polypeptide of SEQ ID NO: 2". This also applies to the expression "consisting of a base sequence".
[0023]
　In the present invention, "immunity inducing activity" means the ability to activate and expand T cells that respond to cancer cells expressing PDS5A protein. Specifically, the ability of IFN-γ production of cytotoxic T cells and / or helper T cells stimulated with PDS5A protein or a partial polypeptide thereof is higher than that of unstimulated control T cells, PDS5A The cytotoxic activity of cytotoxic T cells stimulated with protein or partial polypeptide thereof with PDS5A protein expressing cancer cells is higher than that of unstimulated control T cells, that PDS5A protein or a partial polypeptide thereof That the stimulated helper T cells potentiate the cytotoxic T cell cytotoxic activity more than that of unstimulated control T cells or that the cytotoxic T cells stimulated with the PDS5A protein or a partial polypeptide thereof That the cells or helper T cells proliferate better than that of the non-stimulated control T cells Taste.
[0024]
　The proliferation of the cells can be confirmed by visual observation, cell counting under a microscope, flow cytometry, the amount of tritium thymidine incorporated into the cells in the medium, and the like. Measurement of the IFN-γ production ability can also be confirmed, for example, using a known ELISPOT assay or the like. Specifically, for example, as described in Examples described later, first, T cells are incubated with a polypeptide to be evaluated for immunity-inducing activity (PDS5A protein or a partial polypeptide thereof in the present invention) and peripheral blood mononuclear T cells are contacted with antigen presenting cells presenting the polypeptide to be evaluated by co-culturing with antigen presenting cells derived from spheres (hereinafter referred to as "PBMCs"). Subsequently, IFN-γ produced from T cells is measured using an antibody specific for IFN-γ. Thereby, the number of immune cells in the T cells can be measured. From this measurement result, immunity induction activity can be evaluated.
[0025]
　In addition, the cytotoxicity can be measured, for example, by co-culturing T cells with a polypeptide to be evaluated for cytotoxicity activity (PDS5A protein or a partial polypeptide thereof in the present invention) and antigen presenting cells derived from PBMC, Can be evaluated by examining whether it exhibits the ability to inhibit proliferation of tumor cells outside or to kill tumor cells (hereinafter referred to as "cytotoxicity activity"). The contact between T cells and antigen presenting cells can be achieved by co-culturing both in a liquid medium, as described below. Measurement of the cytotoxic activity can be carried out by a known method called 51 Cr release assay described, for example, in Int. J. Cancer, 58: P 317, 1994 .
[0026]
　By administering the T cells induced above to tumor-bearing living bodies, the tumor can be reduced or regressed by the cytotoxic activity of the T cells. Therefore, the immunity-inducing activity can also be evaluated as an ability to suppress the proliferation of cancer cells or to reduce or eliminate cancer tissue (tumor) (hereinafter referred to as "antitumor activity").
[0027]
　When the polypeptide is used for therapeutic or prophylactic use of cancer, there is no particular limitation, but it is preferable that the evaluation of immunity induction activity is based on cytotoxic activity or antitumor activity.
[0028]
　As is well known in the art, polypeptides of about 7 amino acid residues or more can include epitopes so that they can exhibit antigenicity and immunogenicity and can have immunity inducing activity, so that the immunity induction It can be used as an agent.
[0029]
　Therefore, the polypeptide of (a) is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 at positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463 , 472 to 498, 533 to 567, 613 to 643, 671 to 735, 737 to 780, 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069 , Or a polypeptide consisting of 7 or more consecutive, preferably 8, 9 or 10 or more consecutive amino acids within the region of positions 1074 to 1215, and having immunity inducing activity. Particularly preferably, the amino acid sequence shown in SEQ ID NO: 2 is located at positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, 533 At positions 567, 613 to 643, 671 to 735, 737 to 780, 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, or 1074 to 1215 With the indicated amino acid sequence.
[0030]
　As a principle of immunity induction by administering a cancer antigen polypeptide, a polypeptide is taken up by an antigen presenting cell and then degraded by peptidase in the cell to become a smaller fragment, and then the fragmented antigen peptide Are presented on the surface of the antigen presenting cells. It is known that cytotoxic T cells and the like recognize antigens presented on the cell surface and selectively kill cancer cells presenting the antigen on the cell surface. It is also possible that helper T cells recognize antigens presented on the surface of antigen presenting cells and promote the induction of cytotoxic T cells that selectively kill cancer cells presenting the antigen on the cell surface Are known. The size of the antigenic polypeptide presented on the surface of the antigen presenting cell is relatively small, about 7 to 30 in number of amino acids. Accordingly, from the viewpoint of presenting it on antigen-presenting cells, the polypeptide of (a) mentioned above is preferably the polypeptide having the amino acid sequence of from 24 to 97, 113 to 132, 134 to 197, 204 To 225 position, 265 to 332 position, 378 to 463 position, 472 to 498 position, 533 to 567 position, 613 to 643 position, 671 to 735 position, 737 to 780 position, 792 to 830 position, 832 to 899 position, 920 To 943, 946 to 993, 1029 to 1069, or 1074 to 1215 in the amino acid sequence shown in SEQ ID NO: 1. About 8 to 30, about 9 to 30, or about 9 to 25 amino acids is sufficient. These relatively small sized polypeptides may be directly displayed on the cell surface on the antigen presenting cells without being taken into the antigen presenting cells.
[0031]
　In addition, polypeptides incorporated into antigen-presenting cells undergo cleavage at random positions by peptidases in the cells to produce various polypeptide fragments, these polypeptide fragments are presented on the antigen presenting cell surface Therefore, the amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence at positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, Such as positions 567, 613 to 643, 671 to 735, 737 to 780, 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, or 1074 to 1215 , Administration of a large-sized polypeptide results in the production of poly Peptide fragment is inevitably arise. Therefore, for induction of immunity via antigen presenting cells, polypeptides of large size can also be used. For example, the number of amino acids may be 30 or more, preferably 40 or more, more preferably 50 or more, further preferably 100 or more.
[0032]
　Furthermore, the polypeptide of the present invention has 8 to 25, preferably 9 to 24, more preferably 9 to 23, having a binding motif with a class I molecule or a class II molecule of MHC (HLA in humans) (BIMAS) HLA Peptide Binding Predictions (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.htmL), which can search for epitope peptides consisting of amino acids, Or SYFPEITHI to screen peptides that can be epitope peptides. More specifically, the amino acid sequence represented by SEQ ID NO: 2 is selected from the group consisting of positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, Positions 533 to 567, 613 to 643, 671 to 735, 737 to 780, 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, or 1074 to 1215 A polypeptide consisting of 7 or more consecutive amino acids within the region of SEQ ID NO: 2. For example, a polypeptide having the polypeptide represented by SEQ ID NO: 3 to 67 or a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 to 67 as a partial sequence and having 10 to 30 amino acid residues . Among these, among the polypeptides comprising as a partial sequence the polypeptide represented by SEQ ID NO: 3 to 67 and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to 67, and having 10 to 30 amino acid residues , The immunity inducing activity of the polypeptide represented by SEQ ID NO: 3 to 67 is due to binding to MHC class I molecule, and the immunity inducing activity of the polypeptide represented by SEQ ID NO: 35 to 67 is due to association with MHC class II molecule .
[0033]
　On the other hand, the above polypeptide (b) is a polypeptide in which one or several amino acid residues of the polypeptide of the above (a) are substituted, deleted and / or added, Is a polypeptide having inducible activity. For example, a polypeptide comprising one to several amino acids deleted, substituted or added in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to 67 can be mentioned.
[0034]
　In the present specification, the "number" in "several" represents an integer of 2 to 10, preferably an integer of 2 to 6, more preferably an integer of 2 to 4, further preferably an integer of 2 or 3.
[0035]
　Generally, modification of one or several amino acids in a given polypeptide is considered not to affect the function of the original polypeptide, in some cases enhancing the desired function of the original polypeptide It is thought that there is even. Indeed, modified peptides composed of amino acid sequences in which one or several amino acid residues have been altered (ie, substituted, deleted, added and / or inserted) as compared to the original amino acid sequence , It is known to retain the biological activity of the original peptide (Mark et al., 1984, Proc Natl Acad Sci USA, 81: 5662-5666, Zoller and Smith, 1982, Nucleic Acids Res. 10: 6487- 6500, Dalbadie-McFarland et al., 1982, Proc Natl Acad Sci USA. 79: 6409-6413). Therefore, the polypeptide of the above (b) can also exhibit immunity inducing activity, so it can be used for preparation of the immunity inducing agent of the present invention.
[0036]
　The 20 kinds of amino acids constituting the natural protein are neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro) having a low polar side chain, neutral amino acids having a hydrophilic side chain (Asn Those having similar properties such as acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp), Gln, Thr, Ser, Tyr, Cys) , And it is known that the properties of polypeptides often do not change if they are substituted among them. Therefore, in the case of substituting the amino acid residues in the polypeptide of the above (a) of the present invention, substitution between these groups is preferable because it increases the possibility of maintaining immunity inducing activity.
[0037]
　In addition, the polypeptide of (b) above is a polypeptide having the amino acid sequence at positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, 533 to 567 , From 903 to 643, from 671 to 735, from 737 to 780, from 792 to 830, from 832 to 899, from 920 to 943, from 946 to 993, from 1029 to 1069, or from 1074 to 1215 , Preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, or 99.5% or more, and may have immunity inducing activity .
[0038]
　In the present specification, the "identity" of the amino acid sequence (or base sequence) means that both amino acid sequences (or nucleotides) are aligned so that the amino acid residues (or bases) of two amino acid sequences (Or the number of matched bases) is divided by the total number of amino acid residues (or the total number of bases), and the result is expressed as a percentage. At the time of the above alignment, a gap is appropriately inserted into one or both of the two sequences to be compared, if necessary. The alignment of such sequences can be carried out using well-known programs such as BLAST, FASTA, CLUSTAL W, for example. When a gap is inserted, the total number of amino acid residues is the number of residues in which one gap is counted as one amino acid residue. If the total number of amino acid residues counted in this manner is different between the two sequences to be compared, the sequence identity (%) is the total number of amino acid residues of the longer sequence, and the number of identical amino acid residues It is calculated by dividing the number.
[0039]
　When used in the context of cancer therapy or prophylaxis, the polypeptides of the invention should preferably be presented on the surface of cells or exosomes as a complex with each type of HLA. Therefore, it is preferable to select not only an immunity inducing activity but also a peptide having a high binding affinity for each type of HLA. For that purpose, the peptide may be modified by substitution, insertion, deletion, and / or addition of amino acid residues to obtain a modified peptide with improved binding affinity. In addition to naturally presented peptides, the regularity of the sequence of peptides presented by binding to each type of HLA is known (J Immunol, 1994, 152: 3913; Immunogenetics, 1995, 41: 178 ; J Immunol, 1994, 155: 4307) and modifications based on such regularity can be introduced into the immunogenic peptides of the invention. For example, in order to increase the HLA-A24 binding affinity it is desirable to replace the second amino acid from the N-terminus with leucine or methionine and / or to replace the C-terminal amino acid with valine or leucine is there. Therefore, the peptide having the amino acid sequence of SEQ ID NO: 20 to 34, the amino acid second from the N-terminus of these peptides is substituted with leucine or methionine and / or the C-terminal of the amino acid is substituted with valine or leucine Are included in the scope of the present invention.
[0040]
　The substitution can be introduced not only at the position of the terminal amino acid but also at a position where TCR recognition of the peptide is possible. Several studies have demonstrated that amino acid substitutions of peptides are equivalent or superior to the original, including CAP 1, p 53 (264 - 272), Her-2 / neu (369-377), or gp100 (209-217) (Zaremba et al. 1997, Cancer Res. 57: 4570-4577, T. K. Hoffmann et al. 2002, J Immunol. 168 (3): 1338 -47, S.O. Dionne et al. 2003, Cancer Immunol immunother.52: 199-206, and S.O. Dionne et al. 2004, Cancer Immunology, Immunotherapy, 53: 307-314).
[0041]
　In addition to the above modifications, the polypeptides of the invention can also be linked to other substances so long as the resulting ligation polypeptide retains the necessary immunity-inducing activity of the original peptide. Examples of other substances include, but are not limited to, peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. The peptide can include modifications such as glycosylation, side chain oxidation or phosphorylation, provided that the modification does not impair the biological activity of the original peptide. These types of modifications can be made to confer additional functions (eg, targeting and delivery functions) or to stabilize the polypeptide. For example, techniques for introducing D-amino acids, amino acid mimetics or unnatural amino acids to increase the in vivo stability of polypeptides are known in the art and this concept can also be applied to the polypeptides of the invention . Stability of polypeptides can be assayed in several ways. Stability can be tested using various biological media such as peptidase and human plasma and serum (eg Verhoef et al., 1986, Eur J Drug Metab Pharmacokin, 11: 291-302 reference).
[0042]
　Furthermore, the polypeptide of the present invention may be linked to another peptide via a spacer or a linker. Examples of other peptides include, but are not limited to, epitope peptides derived from other polypeptides. Alternatively, two or more polypeptides of the invention may be linked via a spacer or linker. The peptides to be linked via a spacer or a linker may be the same or different from each other. The type of the spacer and the linker is not particularly limited and includes those composed of peptides, more preferably those composed of peptides having one or more cleavage sites that can be cleaved by enzymes such as peptidase, protease and proteasome . Examples of linkers or spacers include, but are not limited to, AAY (P. M. Daftarian et al., J Trans Med, 2007, 5: 26), AAA, NKRK (R. P. M. Sutmuller et al., J Immunol. 2000, 165: 7308-7315) or one to several lysine residues (S. Ota et al., 2002, Can Res. 62: 1471-1476, KS Kawamura et al., 2002 , J Immunol. 168: 5709-5715). The present invention contemplates polypeptides linked to other peptides via spacers or linkers.
[0043]
　Where the polypeptide of the invention comprises cysteine ​​residues, these polypeptides tend to form dimers via disulfide bonds between the SH groups of cysteine ​​residues. Therefore, a dimer of the polypeptide is also included in the polypeptide of the present invention.
[0044]
　The polypeptides of the present invention can be prepared using well-known techniques. For example, it can be synthesized according to a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method) and the like. It can also be synthesized by a conventional method using various kinds of commercially available peptide synthesizers.
[0045]
　Furthermore, a polynucleotide encoding the above-mentioned polypeptide is prepared using a known genetic engineering technique, the polynucleotide is incorporated into an expression vector, introduced into a host cell, and the desired polypeptide To produce the desired polypeptide. When obtaining a polypeptide of interest from a host cell, it may be purified or isolated so as to be substantially free of other native host cell proteins and fragments thereof, or any other chemical substance.
[0046]
　The polynucleotide encoding the polypeptide can be easily prepared by a known genetic engineering technique or an ordinary method using a commercially available nucleic acid synthesizer. For example, a DNA having the nucleotide sequence of SEQ ID NO: 1 is subjected to PCR using a pair of primers designed to amplify the nucleotide sequence shown in SEQ ID NO: 1 using a human chromosome DNA or cDNA library as a template Can be prepared. Reaction conditions for PCR can be appropriately set, for example, a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) , For example, 30 cycles, followed by reaction at 72 ° C. for 1 minute, but it is not limited thereto. Further, based on the information on the nucleotide sequence and amino acid sequence shown in SEQ ID NO: 1, appropriate probes and primers are prepared, and a cDNA library of human or the like is screened using the probe and primer to isolate the desired DNA can do. It is preferable that the cDNA library is prepared from cells, organs or tissues expressing the protein of SEQ ID NO: 2. Procedures such as preparation of the aforementioned probes or primers, construction of cDNA libraries, screening of cDNA libraries, cloning of target genes and the like are known to those skilled in the art and are described, for example, in Green, MR and Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, or Current Protocolin Molecular Biology: www. currentprotocols. can be carried out according to the method described in Com. From the DNA thus obtained, a DNA encoding the polypeptide of the above (a) can be obtained. In addition, since the codon encoding each amino acid is known, the nucleotide sequence of a polynucleotide encoding a specific amino acid sequence can be easily specified. Therefore, since the nucleotide sequence of the polynucleotide encoding the polypeptide of the above (b) can be easily identified, such a polynucleotide may also be synthesized by a conventional method using a commercially available nucleic acid synthesizer .
[0047]
　The host cell may be any cell as long as it can express the polypeptide. Examples of prokaryotic cells include Escherichia coli etc. Examples of eukaryotic cells include mammalian cultured cells such as monkey kidney cells COS 1 and Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, Xenopus egg cells, etc. But are not limited to these.
[0048]
　When prokaryotic cells are used as host cells, expression vectors having origins, promoters, ribosome binding sites, DNA cloning sites, terminators and the like capable of replication in prokaryotic cells are used as expression vectors. Examples of expression vectors for Escherichia coli include pUC system, pBluescript II, pET expression system, pGEX expression system, and the like. When DNA encoding the polypeptide is incorporated into such an expression vector, prokaryotic host cells are transformed with the vector, and the resultant transformant is cultured, the polypeptide encoded by the DNA can be transformed into a prokaryotic Can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.
[0049]
　When eukaryotic cells are used as host cells, expression vectors for eukaryotic cells having promoters, splicing regions, poly (A) addition sites, etc. are used as expression vectors. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 and the like. Similarly to the above, if the DNA encoding the polypeptide is incorporated into such an expression vector, the eukaryotic host cell is transformed with the vector, and the resultant transformant is cultured, then the DNA encodes Can be expressed in a eukaryotic host cell. When pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like is used as an expression vector, a fusion protein with various tags such as His tag, FLAG tag, myc tag HA tag, , The polypeptide can be expressed.
[0050]
　For introduction of the expression vector into the host cell, well-known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method and the like can be used.
[0051]
　In order to isolate and purify a polypeptide of interest from a host cell, known isolation procedures can be used in combination. For example, treatment with a denaturing agent such as urea or surfactant, ultrasonication, enzyme digestion, salting out, solvent fractionation precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, But are not limited to, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.
[0052]
　The polypeptide obtained by the above method includes those which are in the form of a fusion protein with any other protein, as described above. For example, a fusion protein with glutathione-S-transferase (GST) or a His tag can be exemplified. Accordingly, polypeptides in the form of such fusion proteins are also included within the scope of the present invention. Furthermore, the polypeptide expressed in the transformed cell may undergo various modifications within the cell after being translated. Such a post-translationally modified polypeptide also falls within the scope of the present invention as long as it has immunity inducing activity. Examples of such translational modifications include elimination of N-terminal methionine, N-terminal acetylation, glycosylation, limited degradation by intracellular proteases, myristoylation, isoprenylation, phosphorylation and the like.
When
　a polypeptide having an immunity-inducing activity of the present invention or an expression vector containing a gene encoding the polypeptide is administered to a cancer-bearing living body, it is possible to regress a tumor that has already occurred. In addition, by administering the gene encoding the polypeptide or polypeptide having immunity-inducing activity described above to the living body prior to onset of cancer, the occurrence of tumor can be prevented. Therefore, the polypeptide of the present invention or the gene encoding the polypeptide can be an active ingredient of an immunity inducing agent.
[0053]
　Here, the terms "tumor" and "cancer" mean malignant neoplasms and are used interchangeably. In this case, the cancer to be treated is preferably a cancer expressing the PDS5A protein, more preferably leukemia, malignant lymphoma, prostate cancer, liver cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer, large intestine Cancer, stomach cancer, malignant brain tumor, lung cancer or esophageal cancer.
[0054]
　The target animal is preferably a mammal, more preferably a mammal including a primate, a pet animal, a livestock, a competition animal and the like, more preferably a human, a dog or a cat, particularly preferably a human is there.
[0055]
　The target cancer-affected individual (a cancer patient when the individual is a human) is preferably a cancer-affected individual that expresses the PDS5A protein in vivo, and specifically includes cancer described in WO2011 / 027807 It is preferable that the cancer-affected individual is screened by the detection method of cancer. In particular, the expression level of the antibody against the PDS5A protein contained in the sample obtained from the target organism is screened by being larger than the expression level of the antibody contained in the sample obtained from the living body of the healthy individual It is preferably an individual suffering from cancer. Examples of samples to be screened for target cancer-affected individuals include body fluids, tissues, and cells such as blood, serum, plasma, ascites, pleural effusion and the like, but in the case of screening by measuring the expression level of antibodies against PDS5A protein Is preferably serum, plasma, ascites or pleural effusion.
[0056]
　The route of administration of the immunity-inducing agent of the present invention may be oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration or intraarterial administration is preferred. When the immunity-inducing agent is used for the purpose of cancer treatment, it can also be administered to the regional lymph nodes in the vicinity of the tumor to be treated, in order to enhance the anticancer effect. The dosage may be an amount effective for inducing immunity, and may be an amount effective for treatment or prevention of cancer as long as it is used for treatment or prevention of cancer, for example. The effective amount for treating or preventing cancer is appropriately selected according to the size and symptomatology of the tumor, the weight and volume of the target animal, but when the target animal is human, the effective amount per day is usually 0. 0001 to 1000 μg, preferably 0.001 to 1000 μg. This can be administered once or in several divided doses. It is preferable to divide it into several doses per day and administer it every several days or every few months. As specifically shown in the Examples described later, the immunity inducing agent of the present invention can regress already formed tumors. Therefore, since it can exert anti-cancer action also on a small number of cancer cells in the early stage of development, it can prevent the onset and recurrence of cancer if it is used before cancer onset or after cancer treatment. That is, the immunity-inducing agent of the present invention is useful for both treatment and prevention of cancer and can be an active ingredient of a cancer therapeutic or prophylactic agent.
[0057]
　The immunity-inducing agent of the present invention contains the above-mentioned polypeptide of the present invention as an active ingredient, but it may consist of only a single polypeptide or a combination of a plurality of polypeptides. By combining a plurality of polypeptides according to the present invention, it is possible to enhance the immunity induction activity (the induction / activation action of cytotoxic active T cells) possessed by each polypeptide and more effectively achieve treatment or prevention of cancer it can.
[0058]
　The immunity inducing agent of the present invention can also be used in combination with a peptide capable of inducing known cytotoxic T cells. By combining the polypeptides of the present invention, the immunity inducing activity (the induction / activation action of cytotoxic active T cells) possessed by each polypeptide is enhanced, and treatment or prevention of cancer can be more effectively achieved . "Combination" in this case includes administering the immunity-inducing agent of the present invention separately or simultaneously with a peptide capable of inducing known cytotoxic T cells. The term "to be administered separately" as used herein means to administer the immunity-inducing agent of the present invention and a peptide capable of inducing known cytotoxic T cells separately at different time intervals. The order of administration is not limited. On the other hand, "simultaneously administering" means that the immunity-inducing agent of the present invention and a peptide capable of inducing known cytotoxic T cells are premixed and administered in a form integrated, or the immunity-inducing agent And a peptide capable of inducing known cytotoxic T cells in an individual form without any time lag.
[0059]
　The immunity inducing agent of the present invention can be used in combination with other immune enhancing agents capable of enhancing an immunological response in vivo. Other immunopotentiating agents may be contained in the immunity inducing agent of the present invention or may be administered to the patient in combination with the immunity inducing agent of the present invention as a separate composition.
[0060]
　As the above "other immunopotentiating agent", for example, an adjuvant can be mentioned. Adjuvants can enhance the anticancer effect because they can enhance the immunological response by providing antigen reservoir (extracellular or macrophage), activating macrophages and stimulating specific sets of lymphocytes be able to. Therefore, when the immunity-inducing agent of the present invention is used as an active ingredient of a cancer therapeutic or prophylactic agent, the immunity-inducing agent preferably further contains an adjuvant in addition to the polypeptide of the present invention which is the active ingredient. Numerous types of adjuvants are well known in the art and any adjuvant can be used. Specific examples of adjuvants include, but are not limited to, MPL (SmithKline Beecham), the same obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 liposaccharide of Salmonella; QS21 (SmithKline Beecham), purified from Quillja saponaria extract QS-7, QS-17, QS-18 and QS-L1 (So, H.S., et al., 1997, Molecules (Molecules)) described in PCT application WO 96/33739 (SmithKline Beecham) and Freund's incomplete adjuvant; Freund's complete adjuvant; vitamin E; montanide; alum; CpG oligonucleotide Tide (see, for example, Kreig, A. M., et al., 1995, Nature 374: 546-549); various water-in-oil emulsions prepared from poly IC and its derivatives (such as poly ICLC) and biodegradable oils such as squalene and / or tocopherol. Among them, Freund's incomplete adjuvant, montanide, poly IC and derivatives thereof, and CpG oligonucleotide are preferred. The mixing ratio of the adjuvant and the polypeptide is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably about 1: 1. However, the adjuvant is not limited to the above examples, and other adjuvants known in the art can also be used for administering the immunity-inducing agent of the present invention (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Version, 1986). Methods for preparing mixtures or emulsions of immunity inducing agents and adjuvants are well known to those skilled in the art of vaccination.
[0061]
　In addition to the above adjuvants, factors that stimulate the subject's immune response can also be used as the above-mentioned other immune enhancing agent. For example, various cytokines having characteristics of stimulating lymphocytes and antigen presenting cells can be used in combination with the immunity inducing agent of the present invention as an immunopotentiating agent. Numerous cytokines capable of potentiating such immunological responses are known to those skilled in the art, examples of which include interleukin-12 (IL-12), which has been shown to enhance the protective action of the vaccine, But not limited to, GM-CSF, IL-18, interferon α (IFN-α), interferon β (IFN-β), interferon ω (IFN- ω), interferon γ (IFN-γ) and Flt3 ligand It is not done. Such factors can also be used as the above-mentioned immunopotentiating agent, and can be administered to a patient in combination with the immunity-inducing agent of the present invention as contained in the immunity-inducing agent of the present invention or as a separate composition.
The immunity inducing agent of the present invention can be used as an active ingredient of a therapeutic or prophylactic agent for cancer.
[0062]
　A therapeutic or prophylactic agent for cancer may be formulated by appropriately mixing the immunity inducing agent of the present invention with additives such as pharmacologically acceptable carriers, diluents, excipients and the like suitable for each administration mode it can.
[0063]
　The formulation method and usable additives are well known in the field of pharmaceutical preparations and any method and additives can be used. Specific examples of additives include diluents such as physiological buffer; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine and the like; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth and the like ; Lubricants such as magnesium stearate, polyethylene glycol, talc, silica, and the like, but are not limited thereto. Examples of formulations include oral preparations such as tablets, capsules, granules, powders and syrups, parenteral preparations such as inhalants, injections, suppositories and solutions. These preparations can be prepared by generally known manufacturing methods.
Further
　, by bringing the polypeptide and the antigen presenting cell into contact with each other in vitro, the polypeptide can be presented on the antigen presenting cell. That is, the above-mentioned polypeptide (a) or (b) can be used as a treatment agent for antigen presenting cells. Here, as antigen presenting cells, dendritic cells or B cells carrying MHC class I molecules and class II molecules can be preferably used. A variety of MHC class I molecules and class II molecules have been identified and are well known. The MHC molecule in humans is called HLA.
[0064]
　Examples of HLA class I molecules include HLA-A, HLA-B and HLA-C, and more specifically, HLA-A, HLA-B and HLA-C are listed as HLA class I molecules More specifically, HLA-A11, HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA- A11, HLA-A24, HLA- A31, HLA-A 6801, HLA-B 7, HLA-B 8, HLA-B 2705, HLA-B 37, HLA-Cw 0401, HLA-Cw 0602 and the like.
[0065]
　HLA-DRB 1 * 01, HLA-DRB 1 * 03, HLA-DRB 1 * 04, HLA-DRB 1 * 3, HLA-DRB 1 * 04, HLA- HLA-DRB 1 * 15, HLA-DRB 1 * 15, HLA-DRB 1 * 15, HLA-DQA 1, HLA-DQB 1, HLA-DQB 1, HLA-DRB 1 * 10, HLA-DRB 1 * 07, HLA- DPB1.
[0066]
　Dendritic cells or B cells carrying HLA class I or HLA class II molecules can be prepared from blood or the like by well-known methods. For example, dendritic cells are induced from granulocyte macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4) from bone marrow, umbilical cord blood or patient peripheral blood, and tumor- , It is possible to induce tumor-specific dendritic cells.
[0067]
　Administering an effective amount of this dendritic cell can induce a desirable immune response for the treatment of cancer. Bone marrow and umbilical cord blood provided by a healthy person, bone marrow of a patient himself or peripheral blood etc. can be used as cells to be used. Inherent autologous cells of a patient are preferred because they are highly safe and can also be expected to avoid serious side effects. Peripheral blood or bone marrow may be fresh, cryopreserved or cryopreserved. For peripheral blood, whole blood may be cultured, or only white blood cell components may be separated and cultured, but the latter is more efficient and preferred. Furthermore, among the leukocyte components, mononuclear cells may be separated. In addition, when originating from bone marrow or umbilical cord blood, the whole cells constituting the bone marrow may be cultured, or mononuclear cells may be separated and cultured therefrom. Peripheral blood and its white blood cell components and bone marrow cells include mononuclear cells, hematopoietic stem cells or immature dendritic cells and CD4 positive cells, which are the origin of dendritic cells. The cytokine to be used is not limited as long as it is a natural type or genetically modified type as long as it has characteristics confirmed safety and physiological activity, but preferably the quality to be used for medical use is ensured The standard sample is used in the minimum required amount. The concentration of the cytokine to be added is not particularly limited as long as it is a concentration at which dendritic cells are induced, and is usually about 10 to 1000 ng / mL in terms of the total concentration of cytokines, more preferably about 20 to 500 ng / mL. Culturing can be carried out using a well-known medium usually used for culturing leukocytes. The culturing temperature is not particularly limited as long as leukocyte proliferation is possible, but most preferably about 37 ° C., which is human body temperature. In addition, the gas environment during cultivation is not particularly limited as long as leukocyte proliferation is possible, but it is preferably 5% CO 2Is preferably ventilated. Further, the culturing period is not particularly limited as long as the necessary number of cells are induced, but it is usually carried out between 3 days and 2 weeks. Appropriate suitable equipment can be used for the cell separation and culture, but it is preferable that safety is confirmed for medical use, the operation is stable and convenient. In particular, regardless of general containers such as petri dishes, flasks, bottles, etc., it is also possible to use a laminated container, a multistage container, a roller bottle, a spinner type bottle, a bag type incubator, a hollow fiber column, etc. .
[0068]
　The method of contacting the polypeptide and the antigen presenting cell in vitro can be carried out by a well-known method. For example, it can be achieved by culturing antigen-presenting cells in a culture medium containing the polypeptide. The peptide concentration in the medium is not particularly limited, but is usually about 1 to 100 μg / mL, preferably about 5 to 20 μg / mL. The cell density at the time of culturing is not particularly limited, but is usually about 10 3 to 10 7 cells / mL, preferably about 5 × 10 4 to 5 × 10 6 cells / mL. Culturing is preferably carried out in an atmosphere of 37 ° C. and 5% CO 2 according to a conventional method . The length of peptides that antigen presenting cells can present on the surface is usually about 30 amino acid residues at maximum. Therefore, although not particularly limited, when bringing the antigen-presenting cell and the polypeptide into contact with each other in vitro, the polypeptide may be prepared to have a length of 30 amino acid residues or less.
[0069]
　By culturing the antigen presenting cells in the coexistence of the above-mentioned polypeptides, the peptide is taken up by the MHC molecule of the antigen presenting cell and displayed on the surface of the antigen presenting cell. Therefore, using the above polypeptide, isolated antigen presenting cells containing a complex of the polypeptide and MHC molecule can be prepared. Such antigen presenting cells can present the polypeptide to T cells in vivo or in vitro, induce cytotoxic T cells or helper T cells specific for the polypeptide, and proliferate it can.
[0070]
　An antigen-presenting cell comprising the above-described polypeptide and a complex of MHC molecule prepared as described above is brought into contact with T cells in vitro, whereby cytotoxic T cells specific for the polypeptide or Luper T cells can be induced and allowed to proliferate. This can be carried out by co-culturing the antigen presenting cells and T cells in a liquid medium. For example, the antigen presenting cells can be suspended in a liquid medium, placed in a container such as a well of a microplate, and T cells are added thereto for culturing. Although the mixing ratio of antigen presenting cells and T cells during co-culturing is not particularly limited, it is usually about 1: 1 to 1: 100, preferably about 1: 5 to 1: 20 in terms of the ratio of the number of cells. The density of the antigen presenting cells suspended in the liquid medium is not particularly limited, but is usually about 100 to 10,000,000 cells / mL, preferably about 10,000 to 1 million cells / mL. Cocultivation is preferably carried out in an atmosphere at 37 ° C. and 5% CO 2 according to a conventional method . The cultivation time is not particularly limited, but it is usually about 2 days to 3 weeks, preferably about 4 days to 2 weeks. It is also preferred that co-culturing is carried out in the presence of one or more interleukins such as IL-2, IL-6, IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5 to 20 U / mL, the concentration of IL-6 is usually about 500 to 2000 U / mL, the concentration of IL-12 is usually about 5 to 20 ng / mL However, it is not limited to them. The above co-culture may be repeated once or several times with the addition of fresh antigen-presenting cells. For example, the operation of discarding the co-cultured culture supernatant, adding a suspension of fresh antigen-presenting cells, and further co-culturing may be repeated once or several times. Conditions for each co-culture may be the same as above.
[0071]
　By the above co-culture, cytotoxic T cells and helper T cells specific for the polypeptide are induced and proliferated. Thus, the polypeptide can be used to prepare isolated T cells that selectively bind the complex of the polypeptide and the MHC molecule.
[0072]
　As described in Examples described later, the gene (PDS5A gene) encoding the PDS5A protein is a leukemia white blood cell, a malignant lymphoma tissue, a malignant lymphoma cell, a prostate cancer tissue, a prostate cancer cell, a liver cancer tissue, a liver cancer A cancer cell, a breast cancer tissue, a breast cancer cell, a pancreatic cancer tissue, a pancreatic cancer cell, an ovarian cancer tissue, an ovarian cancer cell, a kidney cancer tissue, a kidney cancer cell, a colon cancer tissue, a colon cancer cell, a gastric cancer tissue, a stomach cancer cell, It is specifically expressed in malignant brain tumor cells, lung cancer tissues, lung cancer cells, esophageal cancer tissues, and esophageal cancer cells. Therefore, in these cancer types, PDS5A protein is thought to be present significantly more than normal cells. When a part of the PDS5A protein present in cancer cells is presented to MHC molecules on the surface of cancer cells and the cytotoxic T cells or helper T cells prepared as described above are administered in vivo, As a marker, cytotoxic T cells can damage cancer cells or enhance cytotoxic activity of cytotoxic T cells. In addition, antigen-presenting cells presenting the polypeptide can induce and proliferate cytotoxic T cells and helper T cells specific for the polypeptide also in vivo, so that the antigen presenting cells can be grown By administration into the body, cytotoxic T cells can damage cancer cells or can enhance the cytotoxic activity of cytotoxic T cells. That is, the cytotoxic T cell, helper T cell and antigen presenting cell prepared using the above polypeptide are also useful as a therapeutic or prophylactic agent for cancer similarly to the immunity inducing agent of the present invention.
[0073]
　In the case of administering the above isolated antigen presenting cells or isolated T cells to a living body, in order to avoid an immune response in a living body that attacks these cells as a foreign body, an antigen presentation It is preferable that cells or T cells are prepared using the polypeptide of (a) or (b) as described above.
[0074]
　The route of administration of a therapeutic or prophylactic agent for cancers containing antigen presenting cells or isolated T cells as an active ingredient is preferably parenteral administration such as intravenous administration or intraarterial administration. The dosage is appropriately selected according to symptoms, purpose of administration and the like, but it is generally 1 to 10 trillion, preferably 1 million to 1 billion, and this is administered once every several days or months It is preferable to administer it. The formulation may be, for example, a suspension of the cells in physiological buffered saline, etc., and can be used in combination with other anticancer agents, cytokines, and the like. One or more additives well known in the pharmaceutical field can also be added.

　Also, by expressing the polynucleotide encoding the polypeptide of (a) or (b) in the body of a target animal, immunity induction, that is, antibody production or cytotoxic T cell Can be induced, and the same effect as administration of the polypeptide can be obtained. That is, the immunity-inducing agent of the present invention contains a polynucleotide encoding the polypeptide of (a) or (b) as described above and contains a recombinant vector capable of expressing the polypeptide in vivo as an active ingredient It may be. As shown in Examples described later, recombinant vectors capable of expressing such antigen polypeptides are also called "gene vaccines".
[0075]
　The vector used for producing the gene vaccine is not particularly limited as long as it is a vector that can be expressed in a target animal cell (preferably a mammalian cell), and may be a plasmid vector or a viral vector, which is publicly known in the field of gene vaccines Any vector may be used. As described above, polynucleotides such as DNA and RNA encoding the polypeptide can be easily prepared by a conventional method. Incorporation of the polynucleotide into the vector can also be carried out using methods well known to those skilled in the art.
[0076]
　The administration route of the gene vaccine is preferably a parenteral administration route such as intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration and the like, and the dosage can be appropriately selected depending on the type of antigen , Usually about 0.1 μg to 100 mg, preferably about 1 μg to 10 mg, as the weight of gene vaccine per kg of body weight.
[0077]
　As a method using a viral vector, for example, RNA virus or DNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus or the like, polynucleotide , And infecting the target animal. Among them, a method using retrovirus, adenovirus, adeno-associated virus, vaccinia virus or the like is particularly preferable.
[0078]
　Other methods include direct administration of the expression plasmid intramuscularly (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method, etc. In particular, DNA vaccine method, liposome Method is preferred.
[0079]
　In order to make the gene encoding the polypeptide used in the present invention act as a medicine in practice, there are in vivo methods of directly introducing the gene into the body, a method of collecting a certain kind of cells from the target animal and introducing the gene outside the body into the cell There is an ex vivo method of introducing the cells back into the body, but the in vivo method is more preferable.
[0080]
　When administered by an in vivo method, it can be administered by a suitable route of administration depending on the disease, symptom and the like for therapeutic purposes. For example, it can be administered intravenously, arterially, subcutaneously, intramuscularly and the like. In the case of administration by in vivo method, for example, it may be in the form of a solution or the like, but in general it is considered to be an injection containing DNA encoding the peptide of the present invention which is an active ingredient and the like, Conventional carriers may be added. In the case of liposomes or membrane-fused liposomes (Sendai virus (HVJ) -liposome etc.) containing the DNA, it can be in the form of a liposome preparation such as a suspension agent, a cryoprotectant, a centrifugal concentration frozen agent and the like.
[0081]
　In the present invention, the phrase "the nucleotide sequence shown in SEQ ID NO: 1" encompasses not only the nucleotide sequence actually shown in SEQ ID NO: 1 but also a sequence complementary thereto. Accordingly, when referring to "a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1", a single-stranded polynucleotide having the nucleotide sequence actually shown in SEQ ID NO: 1, its complementary base sequence , And double-stranded polynucleotides comprising them. When a polynucleotide encoding the polypeptide used in the present invention is prepared, any nucleotide sequence is appropriately selected, but those skilled in the art can easily select the nucleotide sequence.
Example
[0082]
　Hereinafter, the present invention will be described more specifically based on examples.
Example 1 Expression Analysis in Each Tissue
(1) Analysis of Expression of PDS5A Gene in Each Cancer Cell Line
　A gene sequence (SEQ ID NO: 1) encoding the amino acid sequence of human PDS5A protein was obtained from Gene Bank. Expression of the obtained gene in various human cell lines was examined by RT-PCR (Reverse Transcription-PCR) method. The reverse transcription reaction was carried out as follows. That is, 50 to 100 mg of each tissue and 5 to 10 × 10 6Total RNA was extracted from the cells using TRIZOL reagent (manufactured by Life Technologies) according to the attached protocol. Using this total RNA, cDNA was synthesized according to the attached protocol with Superscript First-Strand Synthesis System for RT-PCR (manufactured by Life Technologies). For cDNAs of human normal tissues (brain, hippocampus, testis, colon, placenta), gene pool cDNA (manufactured by Life Technologies), QUICK-Clone cDNA (manufactured by Clontech) and Large-Insert cDNA Library (manufactured by Clontech) There was. The PCR reaction was carried out as follows using the obtained gene-specific primers (the nucleotide sequences of the primers are shown in SEQ ID NOs: 68 and 69). That is, 0.25 μL of the sample prepared by the reverse transcription reaction, 2 μM of each of the above primers, 0.2 mM of each dNTP, 0.65 U of ExTaq polymerase (Takara Shuzo) were added with each reagent and attached buffer, Was set to 25 μL, and the cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 30 times using Thermal Cycler (manufactured by BIO RAD). For comparative control, a primer specific to the GAPDH gene which is a housekeeping gene (the nucleotide sequence of the human GAPDH primer is described in SEQ ID NOs: 70 and 71) was also used at the same time.
[0083]
　As a result, as shown in FIG. 1, the human PDS5A gene is expressed in most cancer cell lines, ie leukemia, malignant lymphoma, prostate cancer, liver cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer, colon cancer, gastric cancer, Expression was detected in malignant brain tumor, lung cancer, esophageal cancer.
(2) Expression of PDS5A protein in human cancer tissues (immunohistochemical staining)
　Immunohistochemical staining was performed using 72 cancerous tissues of paraffin-embedded multiple types of cancer tissue arrays (manufactured by BIOMAX). The human cancer tissue array was treated at 60 ° C. for 3 hours, then placed in a dye bottle filled with xylene and replaced with xylene every 5 minutes three times. The same operation was then carried out with ethanol and PBS-T instead of xylene. Human cancer tissue array was placed in a dye bottle filled with 10 mM citrate buffer solution (pH 6.0) containing 0.05% Tween 20, treated at 125 ° C. for 5 minutes, and allowed to stand at room temperature for 40 minutes or more. Excess moisture around the section was wiped off with a Kimwipe, surrounded by DAKOPEN, and an appropriate amount of Peroxidase Block (manufactured by DAKO) was added dropwise. After standing at room temperature for 5 minutes, it was placed in a dye bottle filled with PBS-T, and operation of replacing PBS-T every 5 minutes was performed three times. As a blocking solution, a PBS-T solution containing 10% FBS was placed and allowed to stand at room temperature for 1 hour in a moist chamber. Next, a commercially available rabbit polyclonal antibody (sigma) reacting with PDS5A protein was adjusted to 10 μg / mL with a PBS-T solution containing 5% FBS and the solution was placed in a moist chamber overnight at 4 ° C. . After washing three times with PBS-T for 10 minutes, a suitable amount of Peroxidase Labelled Polymer Conjugated (manufactured by DAKO) was added dropwise, and the mixture was allowed to stand at room temperature for 30 minutes in a moist chamber. After washing three times with PBS-T for 10 minutes, place DAB color developing solution (made by DAKO) on the plate and let stand at room temperature for about 10 minutes, discard the coloring solution and wash it with PBS-T three times for 10 minutes After rinsing with distilled water, it was placed in 70%, 80%, 90%, 95% and 100% ethanol solutions in turn for 1 minute each, and then allowed to stand overnight in xylene. The slide glass was taken out, sealed with Glycergel Mounting Medium (manufactured by DAKO), and observed.
[0084]
　As a result, the PDS5A protein was strongly expressed in most of the cancer, prostate cancer, liver cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer, colon cancer, gastric cancer, malignant brain tumor, lung cancer, and esophageal cancer which were verified.
Example 2 Induction of Peptide Epitope Reactive CD8 Positive T Cells
(1) Prediction of Peptide Motif Binding to HLA-A 0201 and HLA-A 24
　Information on the amino acid sequence of human PDS 5 A protein represented by SEQ ID NO: 2 was obtained from GenBank Obtained. For the prediction of HLA-A0201 and HLA-A24 binding motifs, using a computer prediction program using known BIMAS software (available at http://bimas.dcrt.nih.gov/molbio/hla_bind/), human PDS5A protein , And analyzed 17 kinds of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 3 to 19 which are expected to be able to bind to HLA-A0201 molecule and 17 kinds of polypeptides which are predicted to be capable of binding to HLA-A24 molecule 14 kinds of polypeptides consisting of the amino acid sequences represented by 20 to 34 were selected. All the selected polypeptides were synthesized by Greiner Japan, Inc. for custom peptide synthesis service. The synthesized polypeptide is guaranteed quality by HPLC analysis and mass spectral analysis.
(2) induction of peptide epitope reactive CD8 positive T cells
　Peripheral blood was isolated from a healthy person positive for HLA-A0201, layered on Lymphocyte separation medium (OrganonpTeknika, Durham, NC) and centrifuged at 1,500 rpm for 20 minutes at room temperature. Fractions containing PBMC were collected and washed three times (or more) in cold phosphate buffer to give PBMC. The obtained PBMC were suspended in 20 mL of AIM-V medium (manufactured by Life Technolo- gies) and allowed to adhere to a culture flask (manufactured by Falcon) at 37 ° C. under 5% CO 2 for 2 hours. Nonadherent cells were used for T cell preparation and adherent cells were used to prepare dendritic cells.
[0085]
　Adherent cells were cultured in AIM-V medium in the presence of IL-4 (1000 U / mL) and GM-CSF (1000 U / mL). IL-6 (1000 U / mL, manufactured by Genzyme), IL-1β (10 ng / mL, manufactured by Genzyme) and TNF- α (1000 U / (10 ng / mL, manufactured by Genzyme) was added to the culture medium and cultured for 2 days, and then the obtained non-adherent cell population was used as dendritic cells.
[0086]
　The prepared dendritic cells were suspended in AIM-V medium at a cell density of 1 × 10 6 cells / mL, and the peptide expected to be able to bind to the HLA-A 0201 molecule selected in the above (1) was suspended in 10 μg / mL , And cultured for 4 hours at 37 ° C. and 5% CO 2 using a 96-well plate . After culturing, the cells were irradiated with X-rays (3000 rad), washed with AIM-V medium and cultured in 10% human AB serum (manufactured by Nabi), IL-6 (1000 U / mL) and IL- 12 (10 ng / mL, manufactured by Genzyme ), And 1 × 10 5 cells were added to each well per well of a 24-well plate . The prepared T cell population was added to each well at 1 × 10 6 cells and cultured at 37 ° C. under 5% CO 2 . After 7 days, each culture supernatant was discarded, dendritic cells treated with each peptide obtained in the same manner as above were irradiated with 10% human AB serum (manufactured by Nabi), IL-7 (10 U / mL, (Cell density: 1 × 10 5 cells / mL) containing AIM-V containing IL-2 (10 U / mL, manufactured by Genzyme ), 1 × Each 10 5 cells was added and further cultured. After repeating the same operation four times at 7-day intervals, stimulated T cells were collected and the induction of CD8 positive T cells was confirmed by flow cytometry.
[0087]
　As a negative control, a peptide (SEQ ID NO: 74) which is a sequence outside the scope of the present invention was prepared based on known peptides (SEQ ID NOS: 75 to 83) which bind to HLA-A0201 molecule and Example 5 of WO 2011/027807 The PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 prepared in the above was used as a comparative example and the same treatment as above was carried out.
[0088]
　For peptides that are expected to be capable of binding to HLA-A24 molecule, a peptide epitope was prepared in the same manner as above using dendritic cells and T cell population derived from peripheral blood of HLA-A24 positive healthy person We attempted to induce reactive CD8 positive T cells. As a negative control, the same treatment was carried out using a peptide (SEQ ID NO: 84) which is a sequence outside the scope of the present invention as PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 as a comparative example.
Example 3 Determination of Cytotoxic T Cell Antigen Epitope
(1) IFN-γ Producing Ability
　For each of the T cells induced in Example 2 (2), in order to investigate the specificity to epitope peptides and proteins, Various polypeptides were pulsed into dendritic cells expressing HLA-A0201 molecule. The dendritic cells were prepared by adding each polypeptide in AIM-V medium at a concentration of 10 μg / mL and culturing at 37 ° C. under 5% CO 2 for 4 hours. In addition, various polypeptides include polypeptides represented by the amino acid sequences of SEQ ID NOS: 3 to 19 which are predicted to be capable of binding to HLA-A0201 molecule, negative control polypeptide (SEQ ID NO: 74), HLA-A0201 molecule PDS5A protein consisting of the known polypeptides (SEQ ID NOS: 75 to 83) to be linked and the amino acid sequence represented by SEQ ID NO: 2 was used. 5 × 10 3 T cells were added to 5 × 10 4 dendritic cells after pulling and cultured for 24 hours in a 96-well plate in AIM-V medium containing 10% human AB serum. After the culture, the supernatant was removed and the production amount of IFN-γ was measured by ELISA method.
[0089]
　As a result, dendritic cells pulsed with the polypeptide represented by the amino acid sequence of SEQ ID NOS: 3 to 19, as compared with lanes 1 and 2 using a dendritic cell not pulsed with a polypeptide and a negative control polypeptide Clearly high IFN-γ production was confirmed in lanes 13 to 29 used (FIG. 2). From these results, it was revealed that the peptides of SEQ ID NOS: 3 to 19 are T cell epitope peptides capable of specifically stimulating HLA-A 0201 positive CD8 positive T cells to proliferate and induce IFN-γ production. Furthermore, the production amount of IFNγ using these peptides is equivalent to the known peptide (lanes 4 to 12) of the amino acid sequence represented by SEQ ID NO: 75 to 83 which binds to the HLA-A0201 molecule and the amino acid represented by SEQ ID NO: 2 It was also found to be significantly higher than IFNγ produced from T cells stimulated with the full-length PDS5A protein consisting of the sequence (lane 3). That is, the polypeptides of SEQ ID NOS: 3 to 19 have remarkably high immunity inducing activity as compared with the peptides reported so far. Although the amino acid sequence of the full-length PDS5A protein represented by SEQ ID NO: 2 contains SEQ ID NOS: 3 to 19 having the above immunity-inducing activity, T Production of IFN-γ produced from cells was low. This suggests that the amino acid sequence of the full-length PDS5A protein did not show sufficient immunity inducing activity, since it contains many sequences that suppress immunity inducing activity.
[0090]
　Furthermore, similarly to the above, for the peptide epitope-reactive CD8 positive T cells induced using the polypeptide represented by the amino acid sequence of SEQ ID NOS: 20 to 34 in Example 3 (2), the specificity to the peptide epitope was determined In order to investigate, it was examined by pulsing the full-length PDS5A protein represented by the amino acid sequence of SEQ ID NO: 2, the negative control polypeptide represented by the amino acid sequence of SEQ ID NO: 84, the polypeptide of SEQ ID NO: 20 to 34 (lanes 4 to 18) The amount of IFN-γ produced by T cells against dendritic cells expressing HLA-A24 molecule was measured by ELISA according to the above method.
[0091]
　As a result, it was confirmed that lanes 4 to 8 using dendritic cells pulsed with the polypeptides of SEQ ID NOS: 20 to 34, as compared with lane 1 of the dendritic cells not pulsed with the polypeptide and lane 2 using the negative control polypeptide, 18, significant IFN-γ production was confirmed in the culture supernatant (FIG. 3).
[0092]
　From these results, it was found that the polypeptides of SEQ ID NOS: 20 to 34 are T cell epitope peptides capable of specifically stimulating HLA-A24 positive CD8 positive T cells to proliferate and induce IFN-γ production . Furthermore, it was also found that the production amount of IFNγ using these polypeptides is remarkably higher than IFNγ produced from T cells stimulated with the full-length PDS5A protein represented by the amino acid sequence of SEQ ID NO: 2. For the same reason as above, it is considered that the full-length PDS5A protein did not show sufficient immunity inducing activity.
(2) Evaluation of cytotoxicity
　Next, the polypeptide represented by the amino acid sequence of SEQ ID NOS: 3 to 19 used in the present invention is HLA-A0201 molecule positive for HLA-A0201 molecule on tumor cells expressing human PDS5A protein Or whether CD8 positive T cells stimulated with the polypeptide of the present invention are capable of damaging tumor cells expressing HLA-A0201 positive human PDS5A protein, or are known We investigated whether tumor cells are significantly impaired compared to the peptides (SEQ ID NOS: 75-83) and CD8 positive T cells stimulated with PDS5A protein.
[0093]
　A human glioma (malignant brain tumor) cell line U251 cell, a leukemia cell line Jurkat cell, a liver cancer cell line SK-Hep1, a breast cancer cell line MCF7, a pancreatic cancer cell line Panc1, an ovarian cancer cell line OVCAR 3, kidney cancer cell line A 498, colon cancer cell line HCT 116, gastric cancer cell line KATO 3, and lung cancer cell line NCI-H 522 (purchased from ATCC) in total of 1 × 10 6 cells in a 50 mL centrifuge tube Collected, 100 μCi of chromium 51 was added and incubated at 37 ° C. for 2 hours. Thereafter, the plate was washed three times with an RPMI medium (manufactured by Kibco) containing 10% fetal bovine serum (hereinafter referred to as FBS, manufactured by Kibco), and 1 × 10 3 cells were added per well in a 96-well V bottom plate , 5 × 10 4 polypeptides represented by the amino acid sequence of SEQ ID NOS: 3 to 19 suspended in RPMI medium containing 10% FBS , negative control polypeptide (SEQ ID NO: 74), known peptide (SEQ ID NO: 75 to 83) and HLA-A0201 positive CD8 positive T cells induced by stimulation with the full-length PDS5A protein represented by the amino acid sequence of SEQ ID NO: 2, respectively, and incubated at 37 ° C. under 5% CO 2 And cultured for 4 hours. After incubation, the amount of chromium 51 in the culture supernatant released from the damaged tumor cells was measured to calculate the cytotoxic activity of CD8-positive T cells induced by the stimulation of each polypeptide and protein .
[0094]
　As a result, HLA-A0201-positive CD8 positive T cells induced by polypeptide stimulation represented by the amino acid sequences of SEQ ID NOS: 3 to 19 were so remarkably noticeable as usual for all the 10 types of cells It was found that it has cytotoxic activity. As representative examples, FIGS. 4A and 4B show the results of cytotoxic activity against U251 cells and Jurkat cells, respectively. For CD8 positive T cells (lanes 13 to 29, respectively) stimulated with the polypeptide represented by the amino acid sequence of SEQ ID NOS: 3 to 19, the polypeptides represented by the amino acid sequences of SEQ ID NOS: 75 to 83 (lanes 4 to 12, 12) and CD8 positive T cells (lane 3) stimulated with the full-length PDS5A protein, shows significantly higher cytotoxic activity against U251 cells and Jurkat cells. On the other hand, CD8 positive T cells induced using the negative control polypeptide (lane 2) were similar to Mock (lane 1) and showed no cytotoxic activity. This result indicates that the polypeptides of SEQ ID NOs: 3 to 19 used in the present invention are presented on the HLA-A0201 molecule on tumor cells expressing HLA-A0201 positive and human PDS5A polypeptide, Suggesting that the polypeptide of the invention has the potential to unexpectably induce CD8 positive cytotoxic T cells capable of damaging such tumor cells. In addition, although the amino acid sequences of the full-length PDS5A protein include SEQ ID NOS: 3 to 19, the cytotoxicity is markedly higher than the cytotoxicity activity by CD8 positive T cells stimulated with the polypeptides of SEQ ID NOS: 3 to 19 Weak (lanes 3, 13-29). This is considered to be because T cells having a strong cytotoxic activity could not be induced because the amino acid sequence of the PDS5A protein contains many sequences that suppress immunity inducing activity.
[0095]
　Similarly, the polypeptide of SEQ ID NO: 20 to 34 is one presented on HLA-A24 molecule on tumor cells expressing HLA-A24 positive human PDS5A protein, or stimulated with the polypeptide of the present invention Positive CD8 positive T cells are capable of damaging tumor cells expressing human PDS5A protein with HLA-A24 positivity, or even markedly impaired tumor cells compared to CD8 positive T cells stimulated with PDS5A protein I studied.
[0096]
　A leukemia cell line THP1 which is positive for HLA-A24 and expresses human PDS5A protein, a human creama cell line KNS-42, a liver cancer cell line SK-Hep1, a kidney cancer cell line Caki1, a colon cancer cell line SW480, a gastric cancer cell line KATO3 Chromium 51 was incorporated into a total of 6 species of the polynucleotide of the present invention and purchased from ATCC and induced by stimulation with a polypeptide represented by the amino acid sequence of SEQ ID NOs: 20 to 34, a negative control polypeptide (SEQ ID NO: 84), and full-length PDS5A protein The amount of chromium 51 in the culture supernatant released from the damaged cells was measured when cultured HLA-A24 positive CD8 positive T cells were cultured.
[0097]
　As a result, HLA-A24 positive CD8 positive T cells stimulated with the polypeptide represented by the amino acid sequences of SEQ ID NOS: 20 to 34 were so remarkably noticeable as usual for all the cancer cells used It was found that it has cytotoxic activity. As representative examples, FIGS. 5A and 5B show the results of cytotoxic activity against THP1 cells and SW480 cells, respectively. In CD8 positive T cells (lanes 4 to 18, respectively) stimulated with the polypeptide represented by the amino acid sequence of SEQ ID NOs: 20 to 34, compared with CD8 positive T cells (lane 3) stimulated with the full length PDS5A protein , THP1 cells and SW480 cells. On the other hand, CD8 positive T cells induced using the negative control polypeptide were similar to Mock (lane 1) and showed no cytotoxic activity (lane 2). Thus, SEQ ID NOs: 20-34 are presented on HLA-A24 molecule on cells expressing HLA-A24 positive and human PDS5A protein, the results suggest that the polypeptides of the invention inhibit such HLA- Suggesting the ability to induce CD8 positive cytotoxic T cells capable of damaging cells.
[0098]
　On the other hand, when the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 34 and the full-length PDS5A protein consisting of the amino acid sequence represented by SEQ ID NO: 2 were exposed to the above 14 types of cancer cells, the cancer cells were completely It did not die. From this fact, it was also confirmed that these polypeptides did not directly kill cancer cells.
[0099]
　As described above, the cytotoxic activity was determined by measuring 5 × 10 4 CD8 positive T cells stimulated with each polypeptide used in the present invention and 1 × 10 3 tumor cells incorporating chromium 51 After incubation for 4 hours, the amount of chromium 51 released into the culture medium after culturing was measured to show cytotoxic activity against each tumor cell (referred to as a target cell) of CD8 positive T cell calculated by the following calculation formula * It is the result.
[0100]
　　　* Formula: cytotoxicity activity (%) = amount of chromium 51 released from target cells when CD8 positive T cells were added ÷ amount of chromium 51 released from target cells plus 1 N hydrochloric acid × 100
Example 4: PDS5A protein Derived peptide epitope reactive CD4 positive T cell induction> To
　predict CD4 positive T cell antigen epitope, the amino acid sequence of human PDS5A protein was analyzed using the SYFPEITHI algorithm (Rammensee's computer prediction program, and HLA class II Thirty-three types of peptides shown in SEQ ID NOS: 35 to 67, which are predicted to be bound peptides, were selected, and all the selected peptides were synthesized as a custom peptide synthesis service by Greiner Japan Co., Ltd.
[0101]
　Peripheral blood was isolated from healthy individuals positive for HLA-DRB1 * 04, layered on Lymphocyte separation medium (manufactured by OrganonpTeknika) and centrifuged at 1,500 rpm for 20 minutes at room temperature. Fractions containing PBMC were collected and washed three times (or more) in cold phosphate buffer to give PBMC. The obtained PBMC was suspended in 20 mL of AIM-V medium (manufactured by Life Technolo- gies) and allowed to adhere to a culture flask (manufactured by Falcon) at 37 ° C. under 5% CO 2 for 2 hours. Nonadherent cells were used for T cell preparation and adherent cells were used to prepare dendritic cells.
[0102]
　On the other hand, adherent cells were cultured in AIM-V medium in the presence of IL-4 (1000 U / mL) and GM-CSF (1000 U / mL). IL-6 (1000 U / mL, manufactured by Genzyme), IL-1β (10 ng / mL, manufactured by Genzyme) and TNF- α (1000 U / (10 ng / mL, manufactured by Genzyme) was added to the AIM-V medium and further cultured for 2 days, and then the obtained non-adherent cell population was used as dendritic cells.
[0103]
　The prepared dendritic cells were suspended in an AIM-V medium at a cell density of 1 × 10 6 cells / mL and each polypeptide of SEQ ID NOS: 35 to 67, a negative control polypeptide (SEQ ID NO: 85) and SEQ ID NO: 2 PDS5A protein consisting of the represented amino acid sequence were each added at a concentration of 10 mg / mL and cultured for 4 hours at 37 ° C. and 5% CO 2 using a 96-well plate . After culture, X-ray irradiation (3000 rad), washing with AIM-V medium, 10% human AB serum (manufactured by Nabi), IL-6 (1000 U / mL) and IL-12 (10 ng / mL, manufactured by Genzyme ), And 1 × 10 5 cells were added to each well per well of a 24-well plate . The prepared T cell population was added to each well at 1 × 10 6 cells and cultured at 37 ° C. under 5% CO 2 . Seven days later, each culture supernatant was discarded, each peptide obtained in the same manner as described above and dendritic cells irradiated with X-ray after treatment with PDS5A protein were suspended in 10% human AB serum (manufactured by Nabi) and IL-2 (10 U / ML, manufactured by Genzyme), 1 × 10 5 cells were added to each well per well of a 24-well plate , and further cultured. After repeating the same operation four times at 7-day intervals, the stimulated T cells were collected and the induction of CD4 positive T cells was confirmed by flow cytometry. As a result, it was confirmed that T cells in each induced hole proliferated.
Example 5: Determination of helper T cell antigen epitope derived from PDS5A protein stimulating HLA-DRB1 * 04 positive CD4 positive T cells>
　PBMC expressing HLA-DRB 1 * 04 molecule was pulsed with various polypeptides in order to investigate the specificity of CD4-positive T cells induced in Example 4 above to each peptide protein. The PBMCs were prepared by adding each polypeptide in AIM-V medium at a concentration of 10 μg / mL and culturing for 4 hours at 37 ° C. and 5% CO 2 . In addition, various polypeptides include polypeptides represented by the amino acid sequences of SEQ ID NOS: 35 to 67, full-length PDS5A protein consisting of the negative control polypeptide (SEQ ID NO: 85) and the amino acid sequence represented by SEQ ID NO: 2 There was. PBMC5 × 10 after the pulse 4 with respect to pieces, 5 × 10 4 was added pieces of CD4-positive T cells were cultured for 24 hours in AIM-V medium containing 10% human AB serum at a 96-well plate. After the culture, the supernatant was removed and the production amount of IFN-γ was measured by ELISA method.
[0104]
　As a result, IFN-γ of 1000 pg / mL or more was produced in the culture supernatant of the wells using PBMC pulsed with the respective peptides of SEQ ID NOs: 35 to 67, respectively. On the other hand, production of IFN-γ was hardly observed in the culture supernatant of the well using only negative control polypeptide and dendritic cells not pulsed with the polypeptide (Mock). Therefore, various polypeptides represented by the amino acid sequences of SEQ ID NOS: 35 to 67 specifically stimulate the growth of HLA-DRB 1 * 04 positive CD4 positive T cells and induce IFN-γ production. T cell epitope peptide It turned out to be. Although the amino acid sequence of the full-length PDS5A protein contains SEQ ID NOs: 35 to 67 having the above immunity-inducing activity, IFN in the culture supernatant of the well using the PBMC cell pulsed with full-length PDS5A protein The amount of - γ produced was extremely small. It is considered that this did not show sufficient immunity inducing activity because the amino acid sequence of the PDS5A protein contains many sequences suppressing the immunity inducing activity.
[0105]
　Next, the polypeptides of SEQ ID NOS: 35 to 67, which have the ability to stimulate growth of HLA-DRB 1 * 04 positive T cells, are processed naturally within the antigen presenting cells from the PDS 5 A protein to generate an epitope presented on HLA-DR It was examined whether or not. A lysate of HEK 293 cells transiently expressing PDS5A protein (purchased from ATCC) was added to immature dendritic cells and digested to matured dendritic cells, and then the polypeptide of SEQ ID NOS: 35 to 67 , Negative control polypeptide and PDS5A protein stimulated T cells were stimulated by the dendritic cells. Peripheral blood was isolated from healthy individuals positive for HLA-DRB1 * 04, layered on Lymphocyte separation medium and centrifuged at 1,500 rpm for 20 minutes at room temperature. The interphase containing PBMC was harvested and washed three times (or more) in cold phosphate buffer to give PBMC. The PBMC thus obtained was suspended in 20 mL of AIM-V medium and allowed to adhere to a culture flask (Falcon) under conditions of 37 ° C. and 5% CO 2 for 2 hours, and adherent cells were cultured in AIM-V medium with IL-4 1000 U / mL) and GM-CSF (1000 U / mL) for 6 days to prepare immature dendritic cells. The above lysate was added to 5 × 10 5 immature dendritic cells and incubated with IL-4 (1000 U / mL), GM-CSF (1000 U / mL), IL-6 (1000 U / mL), IL- 1 β (10 ng / ML) and TNF-α (10 ng / mL) for 2 days in an AIM-V medium. The cultured dendritic cells were irradiated with X-rays (3000 rad), washed with AIM-V medium, suspended in AIM-V medium containing 10% human AB serum, added to each well of 96-well plate at 3.3 × 10 4 each was added. To these, 5 × 10Four polypeptide-negative control polypeptides of SEQ ID NOS: 35 to 67 and T cells stimulated with PDS5A protein were added, and the cells were cultured at 37 ° C and 5% CO 2 for 24 hours. After the culture, the supernatant was removed and the production amount of IFN-γ was measured by ELISA method.
[0106]
　As a result, as shown in FIG. 6, T cells of lanes 4 to 36 stimulated with the polypeptide of SEQ ID NO: 35 to 67 can produce IFN-γ by stimulation of dendritic cells to which PDS5A protein is added all right. On the other hand, production of IFN-γ was hardly observed in lane 2 stimulated with the negative control polypeptide shown in lane 2 and lane 1 not stimulated with the polypeptide. Thus, it was revealed that the polypeptides of SEQ ID NOS: 35-67 are epitopes presented on HLA-DR that PDS5A protein is processed naturally within antigen-presenting cells. In this experiment also, the production amount of IFN-γ was extremely low in lane 3 pulsed with full-length PDS5A protein. It seems that the amino acid sequence of the full-length PDS5A protein did not show sufficient immunity inducing activity, since it contains many sequences suppressing the immunity inducing activity.
Industrial applicability
[0107]
　An immunity inducing agent comprising a polypeptide exhibiting antitumor activity against various cancers of the present invention is useful for treating or preventing cancer or for detecting cancer.
[0108]
　All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.
The scope of the claims
[Claim 1]
　An immunity inducing agent comprising the following (i) or (ii) as an active ingredient.
　(I) at least one polypeptide selected from the group of polypeptides according to (a) or (b) below and having immunity inducing activity,
　　(a) at least one polypeptide selected from the group consisting of 24 to 97 , Positions 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, 533 to 567, 613 to 643, 671 to 735, 737 to 780
　　(B) consisting of consecutive 7 or more amino acids within the region from positions 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, and 1074 to 1215, A polypeptide in which one or several amino acids are deleted, substituted or added in the amino acid sequence of any one of the polypeptides described in (a) above, or
　(ii) A recombinant vector comprising at least one polynucleotide encoding a polypeptide of the present invention and capable of expressing the polypeptide in vivo
[Claim 2]
　The immunity inducing agent according to claim 1, wherein the polypeptide according to (i) binds to MHC class I molecule.
[Claim 3]
　The immunity inducing agent according to claim 2, wherein the polypeptide according to (i) is any one polypeptide selected from the group of polypeptides described in (c) to (e) below.
　　(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 3 to 34
　　(d) a polypeptide in which one to several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide described in (c)
　　(e) a polypeptide comprising the polypeptide according to (c) or (d) as a partial sequence
[Claim 4]
　The immunity inducing agent according to claim 1, wherein the polypeptide according to (i) binds to MHC class II molecule.
[Claim 5]
　The immunity inducing agent according to claim 4, wherein the polypeptide according to (i) is any one polypeptide selected from the group of polypeptides described in (f) to (h) below.
　　(f) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 35 to 67
　　(g) a polypeptide obtained by deleting, substituting or adding one to several amino acids in the amino acid sequence of the polypeptide according to (f)
　　(h) a polypeptide comprising the polypeptide according to (f) or (g) as a partial sequence
[Claim 6]
　The immunity-inducing agent according to any one of claims 1 to 5, which is used as an active ingredient of a therapeutic or prophylactic agent for cancer.
[7]
　7. The immunity inducing agent according to claim 6, wherein the cancer is a cancer expressing PDS5A protein.
[Claim 8]
　The immunity induction according to claim 6 or 7, wherein the cancer is leukemia, malignant lymphoma, prostate cancer, liver cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer, colon cancer, gastric cancer, malignant brain tumor, lung cancer or esophageal cancer Agent.
[Claim 9]
　The immunity inducing agent according to any one of claims 1 to 8, further comprising an immunopotentiating agent.
[Claim 10]
　Any one polypeptide selected from the group of polypeptides described in (a) or (b) below and having immunity inducing activity.
　　(a) an amino acid sequence represented by SEQ ID NO: 2 at positions 24 to 97, 113 to 132, 134 to 197, 204 to 225, 265 to 332, 378 to 463, 472 to 498, In the region of positions 567, 613 to 643, 671 to 735, 737 to 780, 792 to 830, 832 to 899, 920 to 943, 946 to 993, 1029 to 1069, and 1074 to 1215 A polypeptide having an immunity-inducing activity consisting of 7 or more consecutive
　　amino acids in the amino acid sequence of SEQ ID NO: 1 ; (b) a polypeptide having deletion, substitution or addition of one or several amino acids in the amino acid sequence of the polypeptide described in (a) Polypeptide.
[Claim 11]
　11. The polypeptide according to claim 10, which is any one polypeptide selected from the group of polypeptides described in (c) to (e) below.
　　(c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 3 to 34
　　(d) a polypeptide in which one to several amino acids are deleted, substituted or added in the amino acid sequence of the polypeptide described in (c)
　　(e) a polypeptide comprising the polypeptide according to (c) or (d) as a partial sequence
[Claim 12]
　11. The polypeptide according to claim 10, which is any one polypeptide selected from the group of polypeptides described in (f) to (h) below.
　　(f) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 35 to 67
　　(g) a polypeptide obtained by deleting, substituting or adding one to several amino acids in the amino acid sequence of the polypeptide according to (f)
　　(h) a polypeptide comprising the polypeptide according to (f) or (g) as a partial sequence
[Claim 13]
　An isolated antigen-presenting cell comprising a complex of a polypeptide having an immunity-inducing activity and an MHC molecule according to any one of claims 10 to 12.
[Claim 14]
　An isolated T cell that selectively binds a complex of a polypeptide having an immunity-inducing activity and an MHC molecule according to any one of claims 10 to 12.