The name of the invention: the immunity-inducing agent
Technical field
[0001]
　The present invention relates to a novel immunity-inducing agent useful as an active ingredient of cancer therapeutic or prophylactic agent.
Background technique
[0002]
　SCD1 (stearoyl-CoA desaturase 1) protein is a protein to introduce a double bond in the C9-C10 position of saturated fatty acids.
[0003]
　SCD1 protein, associated with the carcinogenesis has been suggested. For example, Non-Patent Documents 1 and 2, liver cancer, esophageal cancer, and expressed in various cancers such as colon cancer rises, inhibiting the function of SCD1 by siRNA or small molecule inhibitor compounds, cancer cell proliferation inhibitory or is, is induced apoptosis was formed tumors is disclosed to be reduced.
[0004]
　On the other hand, in Patent Document 1, SCD1 protein has an immunity-inducing activity against cancer cells, it is disclosed that therefore useful in the treatment or prevention of cancer. However, Patent Document 1, information about peptide binding to MHC molecules have been disclosed.
CITATION
Patent Document
[0005]
Patent Document 1: WO2012 / 157736
Non-Patent Document
[0006]
. Non-Patent Document 1: Igal RA Carcinogenesis Sep; 31 (9): 1509-15 (2010)
Non-Patent Document 2: Chen L. Sci Rep 6, 19665 (2016)..
Summary of the Invention
Problems that the Invention is to Solve
[0007]
　An object of the present invention is to find a new useful polypeptides as an active ingredient of cancer therapeutic or prophylactic agent, to provide a use as immunity-inducing agent of the polypeptide.
[0008]
　Another object of the present invention, the isolated antigen-presenting cell comprising a complex of polypeptides and MHC molecules, and isolated T cells that selectively bind the complex of the polypeptide and the MHC molecule, as well as their cancer to provide a therapeutic or prophylactic agent.
Means for Solving the Problems
[0009]
　Inventors intensively studied, human SCD1 protein malignant lymphoma comprising the amino acid sequence shown in SEQ ID NO: 2, breast cancer, liver cancer, prostate cancer, ovarian cancer, renal cancer, colorectal cancer, stomach cancer, malignant brain tumors, to obtain a finding that is specifically expressed in tissues or cells of esophageal cancer and lung cancer. The partial peptides present in a particular region of the SCD1 protein, are presented by antigen presenting cells, have the ability to activate and proliferate T cells specific to the polypeptide (immunity-inducing activity), and the immunity-inducing activity was found to be useful for treating or preventing cancer. Based on these results, the polypeptide that can become an active ingredient of the immunity-inducing agent for the treatment and / or prevention of cancer, also in contact with antigen-presenting cells and antigen presenting cells contacted with the peptide T found that cells are also useful in the treatment or prevention of cancer, the present invention has been completed.
[0010]
　That is, the present invention is characterized by the following (1) to (12).
(1) the following (a) or is selected from the group of polypeptides according (b), the and the immunity-inducing at least one polypeptide having an activity,
(a) SEQ ID NO: 34 to 50 amino acid sequence shown in 2 position, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, a polypeptide consisting of not less than 7 consecutive amino acids 296 to 332 of the region
according to (b) above (a) any one of the polypeptides 1 to several amino acids are deleted in the amino acid sequence of substitution, insertion or addition polypeptide or of
　at least one comprises said polynucleotide encoding any one of the polypeptides, immunity-inducing agent containing the recombinant vectors, capable of expressing the polypeptide as an active ingredient in vivo.
(2) a polypeptide having the immunity-inducing activity to bind to MHC class I molecules, immunity-inducing agent according to (1).
(3) the is any one of a polypeptide selected from the group of polypeptides according to the polypeptide having an immunity-inducing activity is less than (c) ~ (e), the immunity-inducing agent according to (2).
(c) SEQ ID NO: 3 to an amino acid sequence represented by 36 polypeptide
; (d) (c) 1 - several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide
polypeptide comprising (e) the polypeptide according to (c) or (d) as a partial sequence
(4) a polypeptide having the immunity-inducing activity to bind to MHC class II molecules, immunity-inducing agent according to (1).
(5) the is any one of a polypeptide selected from the group of polypeptides according to the polypeptide having an immunity-inducing activity is less than (f) ~ (h), the immunity-inducing agent according to (4).
(f) comprising the amino acid sequence shown in SEQ ID NO: 37-45 polypeptide
(g) above (f) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide
polypeptide comprising (h) the polypeptide according to (f) or (g) as a partial sequence
is used as an active ingredient (6) cancer therapeutic or prophylactic agents, either (1) to (5) immunity-inducing agent according to.
(7), wherein the cancer is a cancer that expresses SCD1 protein, immunity-inducing agent according to (6).
(8) wherein the cancer is malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, renal cancer, colorectal cancer, stomach cancer, malignant brain tumors, esophageal cancer or lung cancer, immune induction according to (6) or (7) agent.
(9) further comprises an immunopotentiator, the immunity-inducing agent according to any one of (1) to (8).
(10) (1), (3) or (5) isolated antigen presenting cell comprising a complex of polypeptides and MHC molecules having an immunity-inducing activity according to.
[0011]
(11) (1), (3) or (5) to selectively bind to isolate T cells complex of polypeptides and MHC molecules having an immunity-inducing activity according.
(12) the following (a) or (b) is selected from the group of polypeptides according to and any one of the polypeptide having immunity-inducing activity.
(a) 34 ~ 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, successive seven 296-332 positions in the region or more polypeptides having an immunity-inducing activity consisting of the amino acid,
(b) the (a) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide.
(13) below (i) ~ (iv): (i)
　is selected from the group of polypeptides described in the following (a) or (b), and at least one polypeptide having immunity-inducing activity:
　　(a) 34-50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, 296-332 of not less than 7 consecutive amino acids in the region of polypeptide consisting of,
　　(b) the (a) any one of 1 to several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide;
　(ii) the one one of the polypeptides at least one comprises a polynucleotide encoding expressible recombinant vector the polypeptide in vivo;
　(Iii) said any one of the isolated antigen presenting cells containing the complex of polypeptides and MHC molecules; and
　(iv) the specific isolation T cells to said any one of the polypeptides,
selected from the group consisting of that comprises one or more as an active ingredient, therapeutic or preventive agent for cancer.
(14) said at least one polypeptide which is a polypeptide having an immunity-inducing activity selected from the group of polypeptides described in the following (c) ~ (h), treatment or prevention of cancer according to (13) drugs:
(c) consisting of SEQ ID NO: 3-36 amino acid sequence shown in polypeptide;
(d) the (c) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substitution, insertion or the added polypeptide;
(e) the (c) or polypeptide comprising a subsequence of the polypeptide according to (d);
(f) consisting of SEQ ID NO: 37-45 amino acid sequence shown in polypeptide;
(g ;) wherein (1 to several amino acids in the amino acid sequence of a polypeptide according to f) are deleted, substituted, inserted or added polypeptide
polypeptide according to (h) above (f) or (g) polypeptides comprising a partial sequence.
(15) wherein the cancer is a cancer that expresses SCD1 protein, therapeutic or prophylactic agent for cancer according to (13) or (14).
(16) below (i) ~ (iv): (i)
　the following (a) or (b) is selected from the group of polypeptides according to, and at least one polypeptide having immunity-inducing activity:
　　(a) 34 ~ 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, successive seven 296-332 positions in the region or more polypeptides consisting of the amino acid,
　　(b) the (a) any one of 1 to several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide;
　(ii ) wherein at least one comprises a polynucleotide encoding any one of the polypeptides expressible recombinant vector the polypeptide in vivo;
　(iii) a complex of said any one of the polypeptides and MHC molecules isolated antigen presenting cell comprises; and
　(iv) said any one of the polypeptides for the specific isolation T cells,
one or more selected from the group consisting of, a subject in need thereof Comprising administering to an object, a method of treating or preventing cancer.
(17) said at least one polypeptide which is a polypeptide having an immunity-inducing activity selected from the group of polypeptides described in the following (c) ~ (h), the method described in (16):
(c) ; polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 ~ 36
(d) the (c) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide ;
(e) the (c) or polypeptide comprising a subsequence of the polypeptide according to (d);
(f) consisting of SEQ ID NO: 37-45 amino acid sequence shown in polypeptide;
(g) the 1 to several amino acids in the amino acid sequence of a polypeptide according to (f) are deleted, substituted, inserted or added polypeptide;
according to (h) above (f) or (g) polypeptide comprising a polypeptide as a partial sequence.
(18) wherein the cancer is a cancer that expresses SCD1 protein, the method described in (16) or (17).
[0012]
　This description includes the disclosure of the priority document of the present application Japanese Patent Application No. 2016-040364.
Effect of the invention
[0013]
　The present invention, novel immunity-inducing agent useful as an active ingredient of cancer therapeutic or prophylactic agent is provided.
[0014]
　Moreover, as specifically shown in the Examples below, by a polypeptide used in the present invention can induce immune cells to kill cancer cells, it can be reduced or regression of cancer which have already occurred . Moreover, the peptide used in the present invention can enhance the induction of immune cells to kill cancer cells, it can also be reduced or regression of cancer which have already occurred. Thus, the polypeptides of the present invention are useful as an active ingredient of cancer treatment and prophylactic agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
[Figure 1] of the SCD1 gene is a diagram showing the expression patterns in human tumor tissues or cancer cell lines. Reference numeral 1: shows the expression pattern of human SCD1 gene. Reference numeral 2: the expression pattern of the GAPDH gene as a housekeeping gene in humans.
[Figure 2] SEQ ID NO: 3 ~ CD8-positive T cells specific for the polypeptide consisting of the amino acid sequence shown in 23, to produce the IFN-gamma recognizes the complex between the polypeptide and the HLA-A0201 is a diagram illustrating a. Figure, lanes 4-24 on the horizontal axis, by stimulation of dendritic cells polypeptide pulsed represented by the amino acid sequence of SEQ ID NO: 3 ~ 23, IFN-γ production of HLA-A0201-positive CD8-positive T cells show an ability to. Lane 1 shows the result (Mock) for the case of performing the process without addition of the polypeptide, lane 2 is the addition of outside negative control polypeptides of the present invention shown in SEQ ID NO: 46 above processes shows the result of, lane 3 shows the result of the process by addition of full-length SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
[Figure 3] SEQ ID NO: CD8-positive T cells specific for the polypeptide consisting of the amino acid sequence shown in 24-36 are capable of producing IFN-gamma recognizes the complex between the polypeptide and the HLA-A24 is a diagram illustrating a. In the figure, lanes 4-16 on the horizontal axis, by stimulation of dendritic cells polypeptide pulsed represented by the amino acid sequence of SEQ ID NO: 24 ~ 36, IFN-γ production of HLA-A24-positive CD8-positive T cells show an ability to. Lane 1 results for the case of performing the process without addition of a polypeptide indicates (Mock), lane 2 is subjected to the process by adding negative control peptide outside the scope of the present invention shown in SEQ ID NO: 47 It shows the results, and lane 3 shows the result of the process by addition of full-length SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
[Figure 4A] SEQ ID NO: 3 ~ CD8-positive T cells specific for the polypeptide consisting of the amino acid sequence shown in 23 is a diagram showing the cytotoxic activity against cancer cells. Figure, lanes 4 to 24 and the horizontal axis represents the HLA-A0201-positive CD8-positive T cells induced using the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 23, the cytotoxic activity against U251 cells . Lane 1 represents the cytotoxic activity of CD8-positive T cells induced without adding polypeptide (Mock), lane 2 is the CD8-positive T cells induced using the negative control polypeptide (SEQ ID NO: 46) Cell It shows the cytotoxic activity, and lane 3 shows the cytotoxic activity of CD8-positive T cells induced using the full length SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
[Figure 4B] SEQ ID NO: 3 ~ CD8-positive T cells specific for the polypeptide consisting of the amino acid sequence shown in 23 is a diagram showing the cytotoxic activity against cancer cells. In the figure, lanes 4-24 on the horizontal axis, the CD8-positive T cells of HLA-A0201-positive induced with polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 23, for the SK-Hep-1 cells It shows the cytotoxic activity. Lane 1 represents the cytotoxic activity of CD8-positive T cells induced without adding polypeptide (Mock), lane 2 is the CD8-positive T cells induced using the negative control polypeptide (SEQ ID NO: 46) Cell It shows the cytotoxic activity, lane 3 shows the cytotoxic activity of CD8-positive T cells induced using the full length SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
[Figure 5A] SEQ ID NO: 24 ~ CD8-positive T cells specific for the polypeptide consisting of the amino acid sequence shown in 36 is a diagram showing the cytotoxic activity against cancer cells. Lanes 4-16 the horizontal axis shows the cytotoxic activity against SW480 cells CD8-positive T cells stimulated HLA-A24-positive using the polypeptide represented by the amino acid sequence of SEQ ID NO: 24-36. Lane 1 represents the cytotoxic activity of CD8-positive T cells induced without adding polypeptide (Mock), and reference numeral 2 of CD8-positive T cells induced using the negative control polypeptide (SEQ ID NO: 47) It shows the cytotoxic activity, and lane 3 shows the cytotoxic activity of CD8-positive T cells induced using the SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
[Figure 5B] SEQ ID NO: CD8-positive T cells specific for the peptide consisting of the amino acid sequence shown in 24-36 illustrates the cytotoxic activity against cancer cells. Lanes 4-16 on the horizontal axis, the HLA-A24-positive CD8-positive T cells stimulated with the polypeptide represented by the amino acid sequence of SEQ ID NO: 24-36, the cytotoxic activity against ZR-75-1 cells show. Lane 1 represents the cytotoxic activity of CD8-positive T cells induced without adding polypeptide (Mock), and reference numeral 2 of CD8-positive T cells induced using the negative control polypeptide (SEQ ID NO: 47) It shows the cytotoxic activity, and lane 3 shows the cytotoxic activity of CD8-positive T cells induced using the SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
[6] SEQ ID NO: CD4-positive T cells specific for the polypeptide consisting of the amino acid sequence shown in 37-45 may produce IFN-gamma recognizes the complex between the polypeptide and the HLA-DRB1 * 04 It shows that. Lanes 4-12 show the IFN-gamma production ability of HLA-DRB1 * 04-positive CD4 + T cells by stimulation of dendritic cells polypeptide pulsed represented by the amino acid sequence of SEQ ID NO: 37-45. Lane 1 shows the results of the Mock for the case of performing the process without addition of the polypeptide, lane 2 subjected to the above process with the addition of the negative control polypeptide outside the scope of the present invention shown in SEQ ID NO: 48 It shows the results, and lane 3 shows the result of the process by addition of full-length SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2.
DESCRIPTION OF THE INVENTION
[0016]
　
　In the present invention, a "polypeptide" refers to a molecule multiple amino acids are formed by peptide bonds. Not only the number of amino acids is often a polypeptide molecule which constitutes, molecules of amino acid numbers less low molecular weight (oligopeptides) are also included in the polypeptides of the present invention.
[0017]
　The polypeptide constituting the immunity-inducing agent of the present invention is selected from the group of polypeptides described in the following (a) or (b), and include at least one polypeptide having immunity-inducing activity.
(a) in a human SCD1 protein comprising the amino acid sequence shown in SEQ ID NO: 2, 34-50 positions when the initiating methionine and the 1-position (17 amino acids), 69-148 positions (80 amino acids), 178-195 positions ( 18 amino acids), 207-242 position (36 amino acids), 247-280 position (34 amino acids), 296-332 position (37 amino acids) polypeptide consisting of not less than 7 consecutive amino acids in the region of
(b) above ( in the amino acid sequence of a polypeptide according to a), 1 ~ several amino acids are deleted, substituted, inserted or added polypeptide.
[0018]
　In the present invention, the term "comprising the amino acid sequence" means that the amino acid residues are aligned in that order. Thus, for example, the "polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2", having the amino acid sequence of Met Asp Pro Ala ··· (omission) ··· Tyr Lys Ser Gly shown in SEQ ID NO: 2, 359 refers to the size of the polypeptide of the amino acid residues. Further, in the present specification, for example, the "polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2" is often abbreviated as "polypeptide of SEQ ID NO: 2". The same applies to the expression "the nucleotide sequence".
[0019]
　The "immunity-inducing activity" in the present invention means the ability to activation and proliferation of T cells reacting against cancer cells expressing SCD1 protein. Specifically, SCD1 protein or IFN-gamma production capacity of stimulated cytotoxic T cells and / or helper T cells in a partial polypeptide, higher than that of the control T cells not stimulated, SCD1 protein or cytotoxic activity against SCD1 protein-expressing cancer cells stimulated cytotoxic T cells in the partial polypeptide is higher than that of control T cells not stimulated at SCD1 protein or its partial polypeptide stimulated helper T cells and cytotoxic activity of cytotoxic T cells, stimulated to enhance than that of control T cells not, or SCD1 protein or cytotoxic T stimulated with its partial polypeptide cells or helper T cells, means that, well grow than that of the control of T cells not stimulated
[0020]
　Cell growth can be confirmed by visual observation, measurement of the number of cells under a microscope, flow cytometry, uptake, etc. into cells tritiated thymidine in the medium. The measurement of the IFN-gamma production ability, for example, can be confirmed using known ELISPOT assays. Specifically, for example, as described in the Examples below, first, T cells, (SCD1 protein or its partial polypeptide in the present invention) a polypeptide should be evaluated immunity-inducing activity and peripheral blood mononuclear sphere (hereinafter, referred to as "PBMC") by co-cultured with antigen-presenting cells from the T cell is contacted with antigen-presenting cells presenting the polypeptides to be evaluated. Subsequently, the IFN-gamma produced from T cells, measured using an antibody specific for IFN-gamma. This makes it possible to measure the number of immune cells of the T cell. It can be from the measurement results to evaluate the immunity-inducing activity.
[0021]
　Further, after the measurement of the cytotoxic activity, for example, the T cells, which were co-cultured with antigen-presenting cells from and PBMC (SCD1 protein or its partial polypeptide in the present invention) polypeptides to be evaluated cytotoxic activity, biological ability to kill ability or tumor cell inhibits the growth of tumor cells outside (hereinafter, referred to as "cytotoxic activity") it can be evaluated by examining whether showing the. Contact between T cells and antigen presenting cells, as described below, it can be achieved by co-culturing both in a liquid medium. Measurement of cytotoxic activity, for example, Int.J.Cancer, 58: P317,1994 listed in 51 can be carried out by known method called Cr release assay.
[0022]
　By administering T cells induced by the tumor-bearing living body, it can be reduced or regression of tumor by cytotoxic activity of the T cell. Therefore, the immunity-inducing activity is to inhibit the growth of cancer cells, or cancer tissues ability to reduce or eliminate the (tumor) (hereinafter referred to as "antitumor activity") can be evaluated as.
[0023]
　When using the polypeptide for the treatment or prophylactic use of cancer include, but are not limited to, the evaluation of the immunity-inducing activity, it is preferable that the cytotoxic activity or antitumor activity indicators.
[0024]
　As it is known in the art, since the may include an epitope if a polypeptide or about 7 amino acid residues can exert antigenicity and immunogenicity, so may have an immunity-inducing activity, immunity induction of the present invention it can be used as agents.
[0025]
　Thus, the polypeptides of (a) is 34 to 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, 296-332 of less than 7 consecutive in the region of, but preferably having a a polypeptide consisting of 8, 9 or 10 or more contiguous amino acids, and immunity-inducing activity. Particularly preferably, 34 to 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, having 178 to 195 positions, 207-242 positions, 247-280 positions, the amino acid sequence represented by 296-332 of it is intended.
[0026]
　As the principle of immunity induction by administering the cancer antigen polypeptide, the polypeptide is incorporated into antigen-presenting cells, then become smaller fragments undergo degradation by peptidases in said cell, then fragmented antigenic peptides It is presented on the surface of antigen-presenting cells. The antigen presented on the cell surface recognized by cytotoxic T cells and the like, and the antigen is known to go kill cancer cells presenting on the cell surface selectively. Further, to promote the induction of cytotoxic T cells the antigen presented on the surface of an antigen presenting cell helper T cells recognize and kill antigen in selection of cancer cells presenting on the cell surface Are known. The size of the antigenic polypeptides displayed on the surface of antigen-presenting cells are relatively small, about 7 to 30 amino acids number. Therefore, from the viewpoint of presentation on antigen presenting cells, the Polypeptides of (a), 34 ~ 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207 to 242 positions 247 ~ 280 of is preferably continuous about 7 to 30 in the amino acid sequence shown 296 - 332 of. The polypeptide of about 8 to 30, about 9-30, or 9 to a sufficient as long as it is made of a 25 degree acids. Polypeptides of relatively small size, without being incorporated into antigen-presenting cells, the program may be presented on the cell surface on direct antigen presenting cells.
[0027]
　Polypeptides incorporated into the antigen-presenting cell receives the disconnection at random positions by a peptidase in said cell, various polypeptide fragments occurs, these polypeptide fragments are presented on the surface of antigen presenting cells Runode, 34-50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, 296-332 of the large size of the polypeptide as if administered, by degradation of antigen-presenting cells, active polypeptide fragments for immunization induced through the antigen-presenting cells necessarily occur. Thus, immune induction via antigen-presenting cells can also be used to large polypeptides size. For example, the number of amino acids of the polypeptide 30 or more, preferably 40 or more, more preferably 50 or more, more preferably may be 100 or more.
[0028]
　In addition, polypeptides of the present invention is 8 to 25 having a binding motif of the class I molecule or class II molecules below MHC (in humans HLA), preferably from 9 to 24, more preferably 9-23 matching medium that can search an epitope peptide composed of pieces of amino acids, for example, HLA peptide Binding Predictions of Bioinformatics & Molecular Analysis Selection (BIMAS) (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) and collates the SYFPEITHI, it can be obtained by screening peptides which can serve as epitope peptides. Specifically, the polypeptides of the present invention, 34 to 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, 296-332 it is a polypeptide consisting of not less than 7 consecutive amino acids of positions in the region. For example, the polypeptides of the present invention, comprises a polypeptide consisting of SEQ ID NO: 3 ~ polypeptide represented by 45, or SEQ ID NO: 3 to 45 amino acid sequence shown in the partial sequence, and amino acid residues 10 to They include polypeptides that are 30. Among these, polypeptides shown in SEQ ID NO: 3 to 45, or SEQ include ID NOS polypeptide consisting of the amino acid sequence shown in 45 as a partial sequence, and of the polypeptide amino acid residues are 10 to 30 , immunity-inducing activity of the polypeptide shown in SEQ ID NO: 3-36 is due to binding to MHC class I molecules, immunity-inducing activity of the polypeptide shown in SEQ ID NO: 37 through 45 by binding to MHC class II molecules it is intended.
[0029]
　On the other hand, the polypeptide of (b) above, said one or several amino acid residues of the polypeptide by replacing a (a), in deleted, inserted and / or added in the polypeptide, and a polypeptide having an immunity-inducing activity. For example, the polypeptides of the present invention, 1 to several amino acids in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to 45 are deleted, substituted, and insertion or addition polypeptide.
[0030]
　The "number" of "several" herein, an integer of 2 to 10, preferably an integer of 2 to 6, more preferably 2 to 4, more preferably an integer of 2 or 3.
[0031]
　In general, one in the given polypeptide, or modification of a few amino acids is considered to not affect the functionality of the original polypeptide to enhance the desired function of the original polypeptide optionally it is believed that even. In fact, compared to the original amino acid sequence, one or several amino acid residues have been modified (i.e., substitutions, deletions, additions and / or inserted) modified peptide consisting of the 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, since the polypeptide (b) above may exert an immunity-inducing activity, it can be used for preparation of the immunity-inducing agent of the present invention.
[0032]
　Note that 20 types of amino acids constituting natural proteins, neutral amino acids with side chains having low polarity (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids (Asn having hydrophilic side chains , those having Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, similar properties as Trp) can be grouped, it is often the nature of the polypeptide as long as substitution between these unchanged known. Therefore, when replacing the amino acid residues in the polypeptides of (a) of the present invention, by replacing within the same group is preferable because more likely to maintain the immunity-inducing activity.
[0033]
　Also, the polypeptide of (b) above is from 34 to 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, 296-332 of a polypeptide consisting of not less than 7 consecutive amino acids within the region, for example, any of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to 45, 90% or more, preferably 95% or more, more preferably 98% or more, more preferably has a identity of 99% or more or 99.5% or more of the amino acid sequence, and may be a polypeptide having an immunity-inducing activity.
[0034]
　"Identity" of an amino acid sequence herein (or base sequences), amino acid residues (or bases) as much both amino acid sequences to match the two amino acid sequences to be compared (or base sequences) ( or nucleotide sequences) to align the one in which the matched amino acid residues (or the number of matched bases) represents the value obtained by dividing a percentage by total number of amino acid residues (or the total bases). During the alignment, if necessary, to insert the appropriate gaps in one or both of the two sequences to be compared. Alignment of such sequences, e.g. BLAST, it can be performed using FASTA, a well-known program such as CLUSTAL W. If the gap is inserted, the total number of amino acid residues is a number of residues by counting one gap as one amino acid residue. In this way, the total number of amino acid residues counted is, when the different between the two sequences to be compared, sequence identity (%) is the total number of amino acid residues in the longer sequence, matching amino acid residues It is calculated by dividing the number.
[0035]
　When used in the context of cancer treatment or prevention, a polypeptide of the present invention is preferably as a complex with each type of HLA, to be displayed on the surface of cells or exosomes. Thus, the polypeptides of the present invention not only has an immunity-inducing activity, it is preferable to select peptides with high binding affinity for each type of HLA. Therefore, substitution of amino acid residues, insertions, deletions, and / or to modify the peptide by the addition may be modified peptide binding affinity is improved. In addition to peptides that are naturally displayed, (J Immunol since the regularity of the peptide sequences presented by binding to each type of HLA is known, 1994,152: 3913; Immunogenetics, 1995,41: 178 ; J Immunol, 1994,155: 4307), can be introduced into the immunogenic peptides of the present invention modified based on such regularity. For example, in order to enhance the HLA-A24 binding affinity to the second amino acid from the N terminus substituted with leucine or methionine, and / or amino acids it can be desirable to replace by valine or leucine of C-terminal is there. Accordingly, a peptide having the amino acid sequence of SEQ ID NO: 24-36, substituted C-terminal of the second amino acid from the N-terminus of the peptide have been substituted by leucine or methionine, and / or amino acids valine or leucine peptides are are included in the scope of the present invention.
[0036]
　The substitution, not only the location of the terminal amino acids, may also be introduced into the possible locations of the TCR recognition of peptides. Several studies, the amino acid substitutions of peptide has been demonstrated to have or superior immunity-inducing activity is equivalent to the original ones, including, for example CAP1, p53 (264-272), Her -2 / neu (369-377), or there is 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: 307-3 4).
[0037]
　In addition to the above modifications, linked polypeptide the resulting long as it retains the necessary immunity-inducing activity of the original peptide can also be linked to the polypeptide of the present invention with other materials. Examples of other materials include, but are not limited to, peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers. Peptide on the condition that does not impair the biological activity of the original peptide by modification, glycosylation, can contain modifications such as the side chain oxidation or phosphorylation. These types of modifications, additional functions (e.g., targeting function, and delivery function) for imparting, or polypeptide can be performed to stabilize. For example, to increase the in vivo stability of the polypeptide, techniques for introducing D- amino acids, amino acid mimetics or unnatural amino acids are known in the art, it is also possible to apply this concept to a polypeptide of the present invention . Polypeptide stability can be assayed in a number of ways. For example, peptidases, and using a variety of biological media in plasma and serum, etc. of the person, it is possible to test the stability (e.g., Verhoef et al, 1986, Eur J Drug Metab Pharmacokin, 11:. A 291-302 reference).
[0038]
　Furthermore, a polypeptide of the present invention, via a spacer or linker may be linked to another peptide. Examples of other peptides include, but are not limited to, include epitopes peptides derived from other polypeptides. Alternatively, two or more polypeptides of the present invention may be linked via a spacer or linker. Peptides that are linked via a spacer or linker, may be different from each other be the same. Types of spacers and linkers is not particularly limited, those composed of peptides, and more preferably include those composed of a peptide having a peptidase, one or more cleavage sites can be cleaved by enzymes such as proteases and proteasomes It is. 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 of lysine residues (S.Ota et al, 2002, Can Res.62:.. 1471-1476, K.S.Kawamura et al, 2002 , J Immunol.168: 5709-5715), and the like. The present invention, through a spacer or linker assumes a linked polypeptide with other peptides.
[0039]
　When the polypeptide of the present invention contain cysteine ​​residues, these polypeptides have a tendency to form dimers through disulfide bonding between SH groups of cysteine ​​residues. Thus, dimers of polypeptides are also included in the polypeptides of the present invention.
[0040]
　Polypeptides of the present invention can be prepared using well known techniques. For example, Fmoc method (fluorenyl methyloxy carbonyl method) can be synthesized according to the chemical synthesis methods such as tBoc method (t-butyloxycarbonyl method). It can also be synthesized by a conventional method using various commercially available peptide synthesizers.
[0041]
　Further, using known genetic engineering techniques, the polypeptides prepared polynucleotide encoding, and introduced into a host cell incorporating the polynucleotide into an expression vector, the polypeptide of interest in a host cell by producing, it is also possible to obtain the polypeptide of interest. To obtain a polypeptide from a host cell of interest, host cell proteins and fragments thereof other natural or any other chemicals to be substantially free, may be purified or isolated.
[0042]
　The polypeptide encoding polynucleotides, by a conventional method using a known genetic engineering technique or commercially available nucleic acid synthesizer, can be readily prepared. For example, DNA having the nucleotide sequence of SEQ ID NO: 1, PCR is performed using a pair of primers designed to the human chromosome DNA or cDNA library was used as a template, can be amplified nucleotide sequence described in SEQ ID NO: 1 it can be prepared by. The reaction conditions for PCR can be set appropriately, for example, 30 seconds 94 ° C. (denaturation), 30 seconds to 1 minute at 55 ° C. (annealing), 1 cycle reaction process consisting of 2 minutes at 72 ° C. (elongation) as, for example, after 30 cycles, there may be mentioned the conditions to react for 1 minute at 72 ° C., but is not limited thereto. Further, based on the information of the nucleotide sequence and amino acid sequence shown in SEQ ID NO: 1, to prepare a suitable probe or primer, by screening a cDNA library of human or the like using the same, isolating the desired DNA can do. cDNA libraries, cells expressing the protein of SEQ ID NO: 2, is preferably produced from an organ or tissue. Preparation of the probes or primers, construction of cDNA library, screening cDNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Green, M.R. And Sambrook, J., 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. It can be carried out according to the method described in com like. Thus from the obtained DNA as a, can be obtained a DNA encoding a polypeptide of the (a). Moreover, codons encoding each amino acid because it is known, the base sequence of a polynucleotide encoding a specific amino acid sequence can be easily identified. Accordingly, since it is possible to easily identify the base sequence of the polynucleotide encoding the polypeptide of the above (b), such polynucleotides also may be synthesized by a conventional method using a commercially available nucleic acid synthesizer .
[0043]
　As the host cell may be any as long as cells capable of expressing the polypeptide. Examples of prokaryotic cells, such as E. coli, and examples of eukaryotic cells, monkey kidney cells COS1, cultured mammalian cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, Xenopus oocytes, etc. can be mentioned It is, but is not limited thereto.
[0044]
　When using a prokaryotic cell as a host cell, the expression vector replicable origin in prokaryotic cells, a promoter, a ribosome binding site, DNA cloning site, an expression vector having a terminator is used. Expression vectors for E. coli, pUC system, pBluescriptII, pET expression system, pGEX expression systems, and the like can be exemplified. Incorporating DNA encoding the above polypeptide into such an expression vector, after a prokaryotic host cell transformed with the vector, followed by culturing the obtained transformant, prokaryotic polypeptides wherein said DNA encodes it can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.
[0045]
　When using eukaryotic cells as host cells, the expression vector, a promoter, a splicing region, a eukaryotic expression vector having a poly (A) addition site and the like is used. Such expression vectors, pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 and the like. Similar to the above, incorporating a DNA encoding the above polypeptide into such an expression vector, after a eukaryotic host cell with the vector and transformed by culturing the obtained transformant, the DNA encodes the by which polypeptides can be expressed in eukaryotic host cells. pIND / V5-His as an expression vector, in the case of using the pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like, His tag, FLAG tag, myc tag HA tag, a fusion protein obtained by adding GFP and various tags as it can be expressed the polypeptide.
[0046]
　Introduction into the host cell expression vectors, electroporation, calcium phosphate method, liposome method, it is possible to use known methods such as DEAE-dextran method.
[0047]
　In order to isolate and purify a polypeptide of interest from the host cells may be carried out by combining known separation operations. For example treatment with denaturing agents or surfactants such as urea, sonication, enzymatic digestion, salting-out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, Africa Niti chromatography, but reverse phase chromatography, and the like, without limitation.
[0048]
　The polypeptide obtained by the above method, as described above, also include those in the form of a fusion protein with any other protein. For example, a fusion protein of glutathione -S- transferase (GST) or His tag can be exemplified. Thus the polypeptides of the forms of such fusion proteins are also within the scope of the present invention. Further, polypeptides expressed in transformed cells after translation may be subject to various modifications within the cell. Such post-translationally modified polypeptides are also as long as it has an immunity-inducing activity, it is included within the scope of the present invention. Examples of such translational modification, elimination of N-terminal methionine, N-terminal acetylation, glycosylation, limited degradation by an intracellular protease, myristoylation, isoprenylation, such phosphorylation can be exemplified.
[0049]
　
　When administering an expression vector comprising a gene encoding the polypeptide or the polypeptide having immunity-inducing activity of the present invention in tumor-bearing living body, it can cause regression of the tumor has already occurred. Further, it is possible to prevent the development of tumors by administering the gene encoding the polypeptide or polypeptide having an immunity-inducing activity as described above to a living body before the onset of cancer. Thus, the gene encoding the polypeptide or the polypeptide of the present invention may be an active ingredient of the immunity-inducing agent.
[0050]
　Here, the term "tumor" and "cancer" refers to a malignant neoplasm, are used interchangeably. In this case, as the cancer of interest, preferably a cancer expressing SCD1 protein, among others preferably malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, renal cancer, colorectal cancer, stomach cancer, malignant brain tumor, esophageal cancer and lung cancer.
[0051]
　Subject animal is preferably a mammal, more preferably a primate, pet animals, livestock, a mammal including a sport animals such as, more preferably a human, dog or cat, particularly preferably humans is there.
[0052]
　Targeted cancer affected individual (individual cancer patients in the case of humans) is preferably a cancer affected individuals expressing SCD1 protein in vivo, specifically, the cancers described WO2011 / 027807 is preferably a cancer affected individuals to be screened by the method of detecting. In particular, the expression level of antibodies to SCD1 protein contained in a sample obtained from a subject organism, are screened by often compared to the expression level of the antibody contained in the sample obtained from a healthy individual biological it is preferable that the cancer affected individuals. The sample to be subjected to the screening for cancer affected individuals of interest, blood, serum, plasma, ascites, if body fluid pleural effusion, etc., tissue, although cells, and the like, be screened by measuring the expression level of antibodies to SCD1 protein is, serum, plasma, ascites or pleural effusion is preferred.
[0053]
　The route of administration of the immunity-inducing agent of the present invention, even by oral administration, or parenteral administration, but intramuscular, subcutaneous, intravenous, parenteral administration, such as intraarterial administration is preferred. When using the immunity-inducing agent for therapeutic purposes of cancer, to enhance the anti-cancer effect it can also be administered to a regional lymph node in the vicinity of the tumor to be treated. The dosage may be an amount effective to induce immunity, for example, if used for the treatment or prevention of cancer, it may be an amount effective for treating or preventing cancer. Amount effective for treatment or prevention of cancer, tumor size and condition, the subject animal body weight is appropriately selected depending on the volume and the like, when the target animal is a human, an effective amount of usually 1 day is 0. 0001 ~ 1000μg, preferably a 0.001 ~ 1000μg. This can be administered in single or divided doses. Divided into several times per day, preferably administered it a few days or several months every. As specifically shown in the Examples below, the immunity-inducing agent of the present invention can cause regression of tumors that have already been formed. Therefore, since the early development of a small number of cancer cells may exert anticancer effects, the use after cancer development before and cancer treatment, it is possible to prevent the onset or recurrence of cancer. That is, the immunity-inducing agent of the present invention is useful for both the prevention and treatment of cancer can be an active ingredient of cancer treatment or prophylactic agent.
[0054]
　Immunity-inducing agent of the present invention contains as an active ingredient a polypeptide of the present invention described above, may consist of only a single polypeptide may be used in combination of two or more polypeptides. By combining a plurality of polypeptides present invention, the immune-inducing activity of each polypeptide has (induction-activating effect of the cytotoxic activated T cells) is enhanced, it is possible to more effectively achieve the treatment or prevention of cancer it can.
[0055]
　Can also be used in combination with a peptide of the immunity-inducing agent of the present invention can induce known cytotoxic T cells. By combining a polypeptide of the present invention, each polypeptide having immunity-inducing activity (induction-activating effect of the cytotoxic activated T cells) is enhanced, it is possible to more effectively achieve the treatment or prevention of cancer . "Combination" in this case includes separate peptides capable of inducing immunity-inducing agent and known cytotoxic T cell of the present invention, or simultaneously to the administration. The term "separately administering" a peptide capable of inducing immunity-inducing agent and known cytotoxic T cell of the present invention, it refers to the administration separately with a time difference. The order in which the administration does not matter. On the other hand, "concurrently administering" refers be administered in the form obtained by integrating premixed peptides capable of inducing immunity-inducing agent and known cytotoxic T cell of the present invention, or immunity-inducing agent of the present invention and it refers to the administration time difference without a peptide capable of inducing a known cytotoxic T cells in the individual form.
[0056]
　Immunity-inducing agent of the present invention may be used in combination with other immune enhancing agents that may enhance the immunological response in vivo. Other immune enhancing agents may be included in the immunity-inducing agent of the present invention may be administered in combination with the immunity-inducing agent of the present invention as separate compositions to the patient.
[0057]
　Above-mentioned "other immune enhancing agents" may be, for example, an adjuvant. Adjuvants provides reservoir of antigen (extracellularly or within macrophages), activating macrophages and by stimulating certain lymphocytes, because it can enhance the immunological response, to enhance the anti-cancer effect can. Therefore, when using the immunity-inducing agent of the present invention to the active ingredient of the therapeutic or prophylactic agent for cancer, the immunity-inducing agent preferably further comprises an adjuvant in addition to the polypeptide of the active ingredient serving present invention. Many types of adjuvants are well known in the art, it can be used in any adjuvant. Examples of adjuvants include, MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide purification and congeners obtained after acid hydrolysis of Salmonella; QS21 (SmithKline Beecham), pure purified from Quillja saponaria extract QA-21 saponin; PCT application WO96 / 33739 DQS21 described (SmithKline Beecham);. QS-7, QS-17, QS-18 and QS-L1 (So, H.S., et al, 1997, Molecules and cells, 7: 178-186); incomplete Freund's adjuvant; Freund's complete adjuvant; vitamin E; Montanide; alum; CpG Origonukureo Plastid (for example, Kreig, A.M., et al., 1995, Nature374: see 546-549); poly IC and a derivative thereof (poly ICLC, etc.) as well as squalene and / or various water-in-oil emulsions prepared from biodegradable oils such as tocopherol. Among these, Freund's incomplete adjuvant, Montanide, poly IC and derivatives thereof, and CpG oligonucleotides are preferred. The mixing ratio of the adjuvant and polypeptide is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably from about 1: 1. However, the adjuvant is not limited to the above examples, known adjuvants other than the in the art may also be used in the administration of the immunity-inducing agent of the present invention (e.g., Goding, Monoclonal Antibodies: Principles and Practice, second edition, see 1986). Process for the preparation of mixtures or emulsions of immunity-inducing agent and adjuvants is well known to those skilled in the vaccination.
[0058]
　Further, as the other immune-enhancing agents, in addition to the adjuvant, it is also possible to use factors that stimulate the immune response of the subject. For example, it may be used in combination with the immunity-inducing agent of the present invention the various cytokines having a property to stimulate lymphocytes and antigen-presenting cells as an immune enhancer. Such immunological number of cytokines capable enhance response are known to those skilled in the art, examples, interleukin-12, which can enhance the protective effects of the vaccine are shown (IL-12), GM-CSF, IL-18, interferon α (IFN-α), interferon β (IFN-β), interferon ω (IFN-ω), interferon γ (IFN-γ) and Flt3 but ligands include, limited to not. Such factors can also be used as the immunopotentiating agent and can be as be included in the immunity-inducing agent or a separate composition of the present invention in combination with the immunity-inducing agent of the present invention is administered to a patient.
[0059]
　
　immunity-inducing agent of the present invention can be used as an active ingredient of cancer therapeutic or prophylactic agent.
[0060]
　Therapeutic or prophylactic agent for cancer, the immunity-inducing agent of the present invention suitable for each dosage form, the carrier is pharmacologically acceptable, diluent, also be formulated by mixing appropriate additives such as excipients it can.
[0061]
　Formulation methods and available additives are well known in the art of pharmaceutical formulation, it can be used any method and additives. Specific examples of the additives, diluents such as physiological buffer; sugar, lactose, corn starch, calcium phosphate, sorbitol, excipients such as glycine; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth and the like binding agents such as; magnesium stearate, polyethylene glycol, talc, and lubricants such as silica include, but are not limited to. The formulation can include tablets, capsules, granules, powders, oral agents such as syrup, inhalant, injection, suppository, parenteral agent such as solution. These preparations can be produced by production method it is generally known.
[0062]
　
　Further, by contacting the said polypeptide with antigen-presenting cells in vitro, the polypeptide can be presented on antigen-presenting cells. Ie, a polypeptide of the above (a) or (b) may be employed as the processing agent of antigen-presenting cells. Examples of the antigen-presenting cells, may be preferably used dendritic cells or B cells bearing MHC class I and class II molecules. Various MHC class I and class II molecules have been identified and are well known. MHC molecules in humans are referred to as HLA. The HLA class I molecules, HLA-A, HLA-B , there may be mentioned HLA-C, more specifically, HLA-A1, HLA-A0201 , HLA-A0204, HLA-A0205, HLA-A0206, can be exemplified HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, HLA-Cw0602 and the like. The HLA class II molecules, HLA-DR, HLA-DQ , there may be mentioned HLA-DP, and more specifically, HLA-DRB1 * 01, HLA -DRB1 * 03, HLA-DRB1 * 04, HLA- DRB1 * 0405, HLA-DRB1 * 07, HLA-DRB1 * 08, HLA-DRB1 * 11, HLA-DRB1 * 13, HLA-DRB1 * 15, HLA-DRB1 * 15, HLA-DQA1, HLA-DQB1, HLA- DPB1 and the like.
[0063]
　Dendritic cells or B cells bearing MHC class I or MHC class II molecules, can be prepared from blood or the like by known methods. For example, from bone marrow, umbilical cord blood or patient peripheral blood using granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4) induces dendritic cells, tumor-associated peptide on the culture system by adding, it can induce tumor-specific dendritic cells.
[0064]
　The dendritic cells by administering an effective amount, can induce an immune response desired in the treatment of cancer. Used cells, bone marrow or umbilical cord blood provided from healthy individuals, it is possible to use patient himself bone marrow or peripheral blood or the like. The original of autologous cells the patient has a high safety, it is also preferable because it can be expected to avoid serious side effects. Peripheral blood or bone marrow may be either fresh sample, cold storage sample and cryopreserved sample. Peripheral blood may be cultured whole blood, only white blood cell component may be cultured in isolation, but the latter is preferred and efficient. It may be further separated mononuclear among white blood cell component. Further, in the case of bone marrow or umbilical cord blood origin may be cultured whole cells constituting the bone marrow may be cultured by separating therefrom mononuclear. Peripheral blood and their white blood cell component, the bone marrow cells, which contain dendritic cell origin become mononuclear hematopoietic stem cells or immature dendritic cells and CD4-positive cells and the like. Cytokines used is not particularly limited as long as safety and physiological activity have been confirmed properties, native or recombinant type, and the like, but not limited for its production method, preferably the quality to be used in medical is ensured the preparation is used in a necessary minimum amount. Concentration of cytokines to be added is not particularly limited as long as the concentration of the dendritic cells is induced, preferably about 10 ~ 1000 ng / mL in a total concentration of normal cytokine, more preferably about 20 ~ 500ng / mL. Culturing can be carried out using well-known media commonly used for culturing leukocytes. While the culture temperature is not particularly limited as long as the proliferation of white blood cells, and most preferably about 37 ° C. which is human body temperature. Although the gaseous environment in the culture is not particularly limited as long as the proliferation of white blood cells, 5% CO 2It is preferable to vent. Further culture period is not particularly limited as long as a period in which the required number of cells are induced, is usually performed between 3 days to 2 weeks. Equipment to be used for cell separation and culture, can be used as appropriate appropriate, confirmed safety for pharmaceutical use, and it is preferable operation is simple and stable. The particular cell culture device, a petri dish, a flask, regardless generally containers such as bottles, the multilayer container and multistage vessel, roller bottle, spinner type bottle, bag type culturing vessel, may also be used hollow fiber column and the like .
[0065]
　The method itself of contacting the said polypeptide with antigen-presenting cells in vitro may be carried out by well known methods. For example, antigen-presenting cells can be achieved by culturing in a culture medium containing the polypeptide. Peptide concentration in the medium is not particularly limited, usually about 1 ~ 100 [mu] g / mL, preferably about 5 ~ 20μg / mL. Although cell density is not particularly limited in the culture, usually 10 3 ~ 10 7 cells / mL, preferably about × 10 5 4 ~ 5 × 10 6 , about cells / mL. Culture is carried out according to a conventional method, 37 ° C., 5% CO 2 preferably performed in an atmospheric. The length of the peptide antigen-presenting cells can present on the surface is usually up to about 30 amino acid residues. Therefore, although not particularly limited, when contacting the antigen presenting cells with the polypeptide in vitro, it may be prepared the polypeptide of the following lengths 30 amino acid residues.
[0066]
　By culturing antigen-presenting cells in the presence of the polypeptides, peptides are incorporated into MHC molecules of antigen presenting cells and presented on the surface of antigen-presenting cells. Thus, by using the polypeptide, comprising the complex between the polypeptide and the MHC molecule, the isolated antigen-presenting cells can be prepared. Such antigen-presenting cells, in vivo or in vitro, the polypeptide is presented to T cells to induce specific cytotoxic T cells or helper T cells to the polypeptide, it is grown it can.
[0067]
　Are prepared as described above, the antigen-presenting cell comprising a complex of said polypeptide and the MHC molecule, by contacting with T cells in vitro, specific cytotoxic to the polypeptide T cells or to induces helper T cells, it can be grown. This, and the antigen-presenting cells and T cells can be conducted by co-culturing in a liquid medium. For example, antigen-presenting cells suspended in a liquid medium, placed in a container such as wells of a microplate, can be performed by culturing with the addition of T cells to this. The mixing ratio of antigen-presenting cells and T cells upon co-culture is not particularly limited, usually, 1 at a ratio of cell number: 1 to about 1: 100, preferably 1: 5 to 1: about 20. The density of the antigen-presenting cells suspended in the liquid medium is not particularly limited, usually about 100 to 10,000,000 cells / mL, preferably about 10,000 to 1,000,000 cells / mL. Co-culture is carried out according to a conventional method, 37 ° C., 5% CO 2 preferably performed in an atmospheric. Culture time is not particularly limited, usually 2 days to 3 weeks, preferably 4 days to 2 weeks. Moreover, co-culture, IL-2, IL-6 , it is preferably carried out in one or more of the presence of interleukin such as IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5 ~ 20 U / mL, the concentration of IL-6 is usually 500 ~ 2000 U / mL or so, the concentration of IL-12 is usually about 5 ~ 20 ng / mL some, but not limited thereto. The above co-culture may be repeated once or several times by adding fresh antigen-presenting cells. For example, discarded culture supernatant after coculture, an operation of performing further co-culture was added to a suspension of fresh antigen-presenting cells, it may be repeated once or several times. Conditions for each co-culture can be the same as described above.
[0068]
　The co-culture described above, the polypeptide-specific cytotoxic T cells and helper T cells are induced and proliferated. Thus, using the polypeptide, that selectively binds the complex between the polypeptide and the MHC molecule, the isolated T cells can be prepared.
[0069]
　As described in the Examples below, genes (SCD1 gene) encoding the SCD1 protein, malignant lymphoma tissues each, malignant lymphoma cells, breast tissue, breast cancer cells, liver cancer tissues, liver cancer cells, prostate cancer tissue, prostate cancer cells, ovarian cancer tissue, ovarian cancer cells, kidney cancer tissue, kidney cancer cells, colon cancer tissues, colon cancer cells, gastric cancer tissues, stomach cancer cells, malignant brain tumor tissue, malignant brain tumor cells, esophageal cancer tissue, esophageal cancer cells, lung cancer tissue, lung cancer cells, is expressed specifically. Thus, in these cancers is believed that SCD1 protein is present significantly more than normal cells. When a part of the SCD1 protein present in the cancer cells is presented in MHC molecules on the surface of cancer cells, cytotoxic T cells or helper T cells were prepared as described above is administered in vivo, it cytotoxic T cells as a marker can enhance the cytotoxic activity of which or cytotoxic T cytotoxic cancer cells. Furthermore, antigen-presenting cells presenting the polypeptides also induce specific cytotoxic T cells and helper T cells to the polypeptide in vivo, it is possible to grow, the antigen presenting cells live by administering into the body, cytotoxic T cells impairs cancer cells, or cytotoxic activity of cytotoxic T cells can enhance it. That is, the cytotoxic T cells and helper T cells were prepared using the above polypeptide, the antigen presenting cells also similar to the immunity-inducing agent of the present invention is useful as a therapeutic or preventive agent for cancer.
[0070]
　When administering the isolated antigen-presenting cells or isolated T cells as described above to a living body, in order to avoid the immune response in vivo to attack these cells as foreign, antigen presentation taken from the patient to be treated cells or T cells, it is preferable that was prepared using the polypeptide of the as above (a) or (b).
[0071]
　The route of administration of the therapeutic or prophylactic agent for cancer comprising the antigen-presenting cells or isolated T cells as an active ingredient, parenteral administration, such as intravenous or intraarterial administration is preferred. The dose is appropriately selected depending on the symptoms and the purpose of administration, etc., 1 to 10 trillion usually preferably from 100 million to 1 billion, every several days or several months preferably administered. Preparations, for example, the cells may be such as those suspended in physiological buffered saline, it may be used in combination with other anticancer agents and cytokines like. It is also possible to add a known one or more additives in the pharmaceutical field.
[0072]
　
　The above-described (a) or (b) polypeptides also by expressing in the body of the subject animal a polynucleotide encoding inducing immunity, i.e. antibodies production and cytotoxic T cells in the living body It can induce, same effect as administering the polypeptide is obtained. That is, the immunity-inducing agent of the present invention comprises a polynucleotide encoding a polypeptide of the above-mentioned (a) or (b), in vivo those containing an expressible recombinant vector the polypeptide as an active ingredient it may be. As shown in Examples below, such antigenic polypeptides allow expression recombinant vectors are also referred to as "genetic vaccine".
[0073]
　Vector used to produce the gene vaccine is in the target animal cells (preferably mammalian in animal cells) is not particularly limited as long as it is a vector capable of expressing in may be a viral vector in a plasmid vector, known in the field of genetic vaccines You may use any vector. Incidentally, a polynucleotide such as DNA or RNA encoding the polypeptide, as described above, can be easily prepared by a conventional method. Further, incorporation of the polynucleotide into a vector can be carried out using methods well known to those skilled in the art.
[0074]
　The route of administration of the gene vaccine is preferably a parenteral route of administration intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration, etc., the dosage can be appropriately selected depending on the antigen such as the type usually per body weight 1 kg, about 0.1 [mu] g ~ 100 mg by weight of the gene vaccine is preferably about 1 [mu] g ~ 10 mg.
[0075]
　The method according to viral vectors, for example retroviruses, adenoviruses, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, the RNA virus or DNA virus, such as Sindbis virus, a polynucleotide encoding the polypeptide the built-in, a method of infecting the like which in the target animal. In this, retrovirus, adenovirus, adeno-associated virus, a method of using vaccinia virus are particularly preferred.
[0076]
　Other methods include a method of administering an expression plasmid directly into muscle (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method and the like, in particular DNA vaccine method, liposome the law is preferable.
[0077]
　To act as a medicament in practice a gene encoding the polypeptide used in the present invention, a gene in vivo method for introducing into the body directly, and the genes were harvested certain cells outside the target animal to the cell there are introduced ex vivo method of returning the cells into the body, in vivo methods are more preferable.
[0078]
　When administered by in vivo methods it can be administered disease therapeutic purposes, via a suitable administration route depending on the condition and the like. For example, it may be administered via intravenous, intraarterial, subcutaneous, intramuscular, and the like. When administered by in vivo method, for example, but may take dosage forms such as solution, in general, is the form of injection containing DNA encoding the peptide of the present invention as an active ingredient, optionally Te, which conventional carriers may also be added. Also, the liposome or membrane-fused liposomes containing the DNA (Sendai virus (HVJ) - liposome, etc.) in the suspension, frozen drug, may be in the form of liposomal formulation such as centrifugation-concentrated frozen drug.
[0079]
　In the present invention, when the said "base sequence shown in SEQ ID NO: 1" is other actually the indicated nucleotide sequence in SEQ ID NO: 1, sequences complementary thereto encompass. Therefore, when the said "polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1" is a single-stranded polynucleotide having the nucleotide sequence actually shown in SEQ ID NO: 1, its complementary nucleotide sequence single-stranded polynucleotide having, and double-stranded polynucleotide composed of these are included. When preparing a polynucleotide encoding the polypeptide used in the present invention is the selecting any one of the nucleotide sequences can be appropriately and easily the selected by those skilled in the art.
Example
[0080]
　Hereinafter, based on the present invention embodiment will be described more specifically.
[0081]
　 (1) SCD1 gene expression analysis in each cancer cell line
　gene sequence encoding the amino acid sequence of human SCD1 protein (SEQ ID NO: 1) were obtained from Gene Bank. The resulting expression in various human cell lines to gene was examined by RT-PCR (Reverse Transcription-PCR ) method. Reverse transcription was performed as follows. That is, each tissue 50 ~ 100 mg and each cell line 5 ~ 10 × 10 6Total RNA was extracted in accordance with the attached protocol using a number of cells from the TRIZOL reagent (Life Technologies, Inc.). The cDNA was synthesized according to the attached protocol by total RNA Superscript First-Strand Synthesis System for RT-PCR using the (Life Technologies, Inc.). cDNA of human normal tissues (brain, hippocampus, testis, colon and placenta), (manufactured by Life Technologies, Inc.) Gene Pool cDNA, use the QUICK-Clone cDNA (Clontech) and Large-Insert cDNA Library (manufactured by Clontech) It had. PCR reactions obtained gene-specific primer (base sequence of the primer is set forth in SEQ ID NO: 49 and 50) were carried out as follows using. That is, the sample 0.25μL prepared by reverse transcription reaction, the above primers each 2 [mu] M, of each dNTP 0.2 mM, a buffer attached with the reagent so that ExTaq polymerase 0.65u (Takara Shuzo) was added, the total amount It was a 25 [mu] L, using a Thermal Cycler (BIO manufactured RAD Co.), 94 ° C. -30 seconds, 55 ° C. -30 seconds, and 72 ° C. -1 minute cycle was repeated 30 times. For comparison, (SEQ ID NO: 51 and 52 nucleotide sequence of human GAPDH primer) primers specific for GAPDH gene is a housekeeping gene were used simultaneously.
[0082]
　As a result, as shown in FIG. 1, the human SCD1 gene, the majority of cancer cell lines, i.e. malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, renal cancer, colorectal cancer, stomach cancer, malignant brain tumors, esophageal cancer and expression in lung cancer was detected.
[0083]
　(2) SCD1 protein expression in human cancer tissues (immunohistochemical staining)
　With cancer tissue 72 specimens many types of cancer tissue arrays paraffin-embedded (BIOMAX Co.), was subjected to immunohistochemical staining. After human cancer tissue array 3 hours at 60 ° C. was repeated three times an operation to replace the xylene every 5 minutes taking into staining jars filled with xylene. Then the same procedure was carried out with ethanol and PBS-T instead of xylene. Put human cancer tissue array Senshokubin filled 10mM citrate buffer (pH 6.0) containing 0.05% Tween20, after 5 min treatment with 125 ° C., and allowed to stand over 40 minutes at room temperature. Wiping the excess water of the surrounding sections with Kimwipe, enclosed in DAKOPEN, it was dropped onto the Peroxidase Block (DAKO Corporation). 5 minutes standing at room temperature, was repeated three times an operation to replace the PBS-T every 5 minutes taking into staining jars filled with PBS-T. As a blocking solution, placing the PBS-T solution containing 10% FBS, allowed to stand 1 hour at room temperature in moist chamber. Then a commercial rabbit polyclonal antibodies reactive to SCD1 protein (sigma, Inc.), placing the solution prepared in 10 [mu] g / mL in PBS-T solution containing 5% FBS, allowed to stand overnight at 4 ° C. in moist chamber . After PBS-T for 10 minutes 3 times wash, Peroxidase Labeled Polymer Conjugated (manufactured by DAKO) was dropped onto and allowed to stand for 30 minutes at room temperature moist chamber. After 3 washes for 10 minutes with PBS-T, topped with DAB color development solution (DAKO Corporation), it was allowed to stand for about 10 minutes at room temperature, discard the coloring solution, washed three times 10 minutes with PBS-T after, rinsed with distilled water, 70%, 80%, 90%, 95%, was placed in each 1 minute in order to 100% of each ethanol solution and allowed to stand overnight in xylene. Removed slide glass, after encapsulation with Glycergel Mounting Medium (DAKO Corporation), was observed.
[0084]
　As a result, most of the cancer SCD1 protein was verified, malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colon cancer, stomach cancer, malignant brain tumors, is a strong expression in esophageal cancer and lung cancer was observed.
[0085]
　 (1) HLA-A0201 and HLA-A24 to the prediction of peptide motifs that bind
　information of the human SCD1 protein amino acid sequence of represented by SEQ ID NO: 2 from GenBank Obtained. HLA-A0201 and HLA-A24 binding for motif prediction, known BIMAS software person using computer prediction program using (available at http://bimas.dcrt.nih.gov/molbio/hla_bind/) SCD1 protein analyzing the amino acid sequence, HLA-A0201 and polypeptide 21 kinds consisting of the amino acid sequence represented by SEQ ID NO: 3 to 23 expected to be capable of binding to the molecule, SEQ ID NO: expected to be capable of binding to HLA-A24 molecules It was selected polypeptide 13 kinds consisting of the amino acid sequence represented by 24-36. All of the polypeptides selected, was synthesized ask the custom peptide synthesis services of Greiner Japan. Incidentally, the synthesized polypeptide is one of guaranteed quality by HPLC analysis and mass spectrometry.
[0086]
　(2) Induction of peptide epitope-reactive CD8-positive T cells
　Peripheral blood was separated from healthy individuals HLA-A0201-positive, Lymphocyte separation medium (OrganonpTeknika, Durham , NC) 20 minutes at room temperature 1,500rpm and overlaid and centrifuged. Fractions were collected containing the PBMC, cold phosphate buffer three times (or more) and washed to give PBMC. The resulting PBMC were suspended in AIM-V medium (Life Technololgies Co.) 20 mL, 37 ° C. in a culture flask (manufactured by Falcon), 5% CO 2 and allowed to adhere for 2 hours under the conditions of. Nonadherent cells are used for T cell preparation, adherent cells were used to prepare the dendritic cells.
[0087]
　Adherent cells were cultured in the presence of IL-4 in AIM-V medium (1000 U / mL) and GM-CSF (1000U / mL). IL-4 after 6 days (1000U / mL), GM-CSF (1000U / mL), IL-6 (1000U / mL, manufactured by Genzyme Corporation), IL-1β (10ng / mL, manufactured by Genzyme Corporation), and TNF-alpha (10 ng / mL, manufactured by Genzyme Corporation) was replaced with AIM-V medium supplemented with were cultured for another 2 days, non-adherent cell population obtained was used as dendritic cells.
[0088]
　The prepared dendritic cells 1 × 10 in AIM-V medium 6 were suspended at a cell density of cells / mL, peptide 10 [mu] g / mL expected to be capable of binding to HLA-A0201 molecules selected in (1) It was added at a concentration, 37 ° C. using a 96-well plate, 5% CO 2 for 4 hours at conditions. After culturing, and X-ray irradiation (3000 rad), washed with AIM-V medium, (manufactured by Nabi Co.) 10% human AB serum, IL-6 (1000U / mL ) and IL-12 (10ng / mL, Genzyme Corp. ) were suspended in AIM-V medium containing, respectively 1 × 10 per 24-well plate 1 well 5 was added in the cells. Further T cell population, respectively per well 1 × 10 prepared 6 cells were added, 37 ° C., 5% CO 2 and cultured under the conditions of. After 7 days, discarded each culture supernatant, the irradiation dendritic cells with 10% human AB serum after treatment X-rays at each peptide was obtained by the same procedure (Nabi Inc.), IL-7 (10U / mL, Genzyme Corp.) and IL-2 (10U / mL, were suspended in AIM-V medium containing manufactured Genzyme Corp.) (cell density: 1 × 10 5 cells / mL), respectively 1 × per 24 well plate 1 well 10 5 was added in the cells were further cultured. The stimulated T cells after repeated 4 times to 7 days every other similar operations were recovered and confirmed the induction of CD8-positive T cells by flow cytometry.
[0089]
　In Comparative as a negative control, the SCD1 protein consisting of a sequence outside the range of peptides (SEQ ID NO: 46) and WO2012 / 157736 Example 3 amino acid sequence of SEQ ID NO: 2 prepared on the basis of the present invention embodiment use as was subjected to the same processing as described above.
[0090]
　As for the peptides expected to be capable of binding to HLA-A24 molecule, in the same manner as described above using the dendritic cells and T cell population derived from peripheral blood of healthy donors of HLA-A24-positive, peptide epitope We tried the induction of reactive CD8-positive T cells. As a negative control, the range of the array in which the peptides of the present invention (SEQ ID NO: 47), was used as a comparative example SCD1 protein consisting of the amino acid sequence of the SEQ ID NO: 2 was subjected to the same treatment.
[0091]
　
　(1) IFN-gamma Sanseino
　for each T cells induced in Example 2 (2), in order to investigate the specificity for the epitope peptides and proteins, various polypeptides were pulsed dendritic cells expressing HLA-A0201 molecules. The dendritic cells were added to each polypeptide in AIM-V medium at a concentration of 10 [mu] g / mL, 37 ° C., 5% CO 2 was prepared by 4-hour culture under the conditions of. Table Moreover, the various polypeptides, in HLA-A0201 the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 23 expected to be capable of binding to the molecule, negative control polypeptide (SEQ ID NO: 46) and SEQ ID NO: 2 using SCD1 protein comprising the amino acid sequence. Dendritic cells 5 × 10 after the pulse 4 with respect to pieces, 5 × 10 3 was added to one T cells were cultured for 24 hours in AIM-V medium containing 10% human AB serum at a 96-well plate. Taking supernatant after incubation was measured by ELISA method production of IFN-gamma.
[0092]
　As a result, compared to lanes 1 and 2 with dendritic cells and negative control polypeptides not pulsed with polypeptide, the dendritic cells pulsed with a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 23 in lanes 4-24 using clearly higher IFN-gamma production was observed (Figure 2). From this result, the peptide of SEQ ID NO: 3 through 23 specifically grown stimulate HLA-A0201-positive CD8-positive T cells were found to be T-cell epitope peptide having the ability to induce IFN-gamma production. Furthermore, production of IFN-gamma using these peptides are significantly higher than the IFN-gamma produced from T cells stimulated with full-length SCD1 protein comprising the amino acid sequence represented by SEQ ID NO: 2 (lane 3) it was also found. That is, the polypeptide of SEQ ID NO: 3 to 23 show that it has a significantly higher immunity-inducing activity. Further, in the amino acid sequence of the full-length SCD1 protein represented by SEQ ID NO: 2 were stimulated with the immunity-inducing activity despite contains SEQ ID NO: 3 to 23 with SEQ ID NO: 2 full-length SCD1 protein T production of IFN-gamma produced from the cell was low. This is in the amino acid sequence of the full-length SCD1 protein, since it contains many suppress SEQ an immunity-inducing activity is believed that did not show sufficient immunity-inducing activity.
[0093]
　Further, similarly to the above, for Example 3 peptide epitopes reactive CD8-positive T cells induced using the polypeptide represented by the amino acid sequence of SEQ ID NO: 24-36 in (2), the specificity for a peptide epitope to investigate, SEQ ID NO: 24-36 polypeptide (lanes 4-16), negative control polypeptide represented by the amino acid sequence of SEQ ID NO: 47, pulsed with the full-length SCD1 protein represented by the amino acid sequence of SEQ ID NO: 2, for dendritic cells expressing HLA-A24 molecules, the production of IFN-gamma T cells was measured by the ELISA according to the above method.
[0094]
　As a result, as compared to lane 2 with lanes 1 and negative control polypeptides of dendritic cells not pulsed with the polypeptides, lane 4 with dendritic cells pulsed with a polypeptide of SEQ ID NO: 24-36 ~ in supernatants in 16 cultures, significant IFN-gamma production was observed (Fig. 3).
[0095]
　From this result, the polypeptide of SEQ ID NO: 24-36 are specifically grown stimulate HLA-A24-positive CD8-positive T cells were found to be T-cell epitope peptide having the ability to induce IFN-gamma production . Furthermore, production of IFN-gamma using these polypeptides has also been found that significantly higher than IFN-gamma produced from T cells stimulated with full-length SCD1 protein represented by the amino acid sequence of SEQ ID NO: 2. The full length SCD1 protein by the same reason as described above, considered did not show sufficient immunity-inducing activity.
[0096]
　(2) cytotoxic Evaluation
　Next, a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 to 23 for use in the present invention, HLA-A0201 molecules on tumor cells expressing human SCD1 protein HLA-A0201-positive or those presented above, also it can be CD8-positive T cells stimulated with a polypeptide of the present invention is impaired tumor cells expressing human SCD1 protein HLA-A0201-positive, more SCD1 protein It was examined whether significantly impaired tumor cells in compared to stimulated CD8-positive T cells.
[0097]
　Human glioma (malignant brain tumor) cell line U251 cells expressing human SCD1 protein have been identified, leukemia cell lines THP1 liver cancer cell line SK-Hep-1, the breast cancer cell lines MCF7, ovarian cancer cell line OVCAR3, renal cancer cell lines A498, colorectal cancer cell lines HCT116, stomach cancer cell lines AGS, and lung cancer cell lines NCI-H522, respectively 10 cell lines (JCRB, RIKEN and purchased from ATCC) 6 collected in a centrifuge tube pieces 50mL volumes of chromium 100μCi 51 was added and incubated for 2 hours at 37 ° C.. Then (hereinafter referred to as FBS, Kibuko Co.) 10% fetal calf serum were washed 3 times with RPMI containing medium (Kibuko Co.), 10 per 96-well V-bottom plate 1 well 3 was added portionwise, still this 10% suspended 5 × 10 in RPMI medium containing of FBS 4 Table polypeptide, negative control polypeptide (SEQ ID NO: 46) and amino acid sequence of SEQ ID NO: 2 represented by the amino acid sequence of consecutive nucleotides 3 to 23 It was added CD8-positive T cells induced HLA-A0201-positive stimulation with full-length SCD1 proteins respectively, 37 ° C., 5% CO 2 for 4 hours at conditions. After incubation, by measuring the amount of chromium 51 in the culture supernatant released from tumor cells damaged it was calculated cytotoxic activity of CD8-positive T cells induced by stimulation of the polypeptides and proteins .
[0098]
　As a result, CD8 + T cells have been HLA-A0201-positive induced polypeptides stimulus represented by the amino acid sequence of SEQ ID NO: 3 to 23 were found to have a significant cytotoxic activity against all the cells. As a typical example, in Figure 4A, and 4B, shows the results of the cytotoxic activity against each U251 cells and SK-Hep-1 cells. Compared to CD8-positive T cells stimulated with a polypeptide represented by the amino acid sequence of SEQ ID NO: 3 ~ 23 CD8-positive T cells stimulated with (lanes 4-24), the full-length SCD1 protein (lane 3), It shows a remarkably high cytotoxic activity against U251 cells and SK-Hep-1 cells. On the other hand, CD8-positive T cells induced using the negative control polypeptide (lane 2) are comparable with Mock (lane 1) showed no cytotoxic activity. This result is for the polypeptide of SEQ ID NO: 3 to 23 for use in the present invention are presented on HLA-A0201 molecules on tumor cells expressing human SCD1 polypeptide in HLA-A0201-positive, yet the polypeptides of the invention, suggest that the ability to induce CD8-positive cytotoxic T cells capable of disorders such tumor cells. Moreover, despite the in the amino acid sequence of the full-length SCD1 protein contains SEQ ID NO: 3 to 23, significantly than cytotoxic activity of CD8-positive T cells stimulated with a polypeptide of SEQ ID NO: 3 to 23 It was weak (lanes 3, 4 to 24). This is considered because it is in the amino acid sequence of SCD1 protein is rich in suppressing SEQ an immunity-inducing activity, for T cells having strong cytotoxic activity could not be induced.
[0099]
　Similarly, a polypeptide of SEQ ID NO: 24-36 is, HLA-A24 positive or those presented on HLA-A24 molecules on the tumor cells expressing human SCD1 protein, also stimulated with a polypeptide of the present invention or CD8 + T cells can impair tumor cells expressing human SCD1 protein HLA-A24-positive, significantly impaired tumor cells more as compared to CD8-positive T cells stimulated with SCD1 proteins It was investigated whether the to.
[0100]
　Expressing human SCD1 protein HLA-A24-positive human glioma cell lines KNS-42, liver cancer cell line SK-Hep1, renal cancer cell lines Caki1, colon cancer cell line SW480, gastric cancer cell line MKN45, prostate cancer cell line PC3 , breast cancer cell lines ZR75-1 chromium 51 incorporated into (JCRB, RIKEN and purchased from ATCC), polypeptide represented by the amino acid sequence of SEQ ID NO: 24-36, negative control polypeptide (SEQ ID NO: 47), and when cultured with CD8-positive T cells induced HLA-A24-positive stimulation with full-length SCD1 protein, to determine the amount of chromium 51 in the culture supernatant released from the damaged cells.
[0101]
　As a result, CD8-positive T cells in HLA-A24-positive stimulated with a polypeptide represented by the amino acid sequence of SEQ ID NO: 24-36 is typically significantly enough not have been predicted in to all cancer cells with it was found to have a such cytotoxic activity. Representative examples shows the results of cytotoxic activity against each SW480 cells, and ZR75-1 cells in FIGS. 5A and 5B. In CD8 + T cells stimulated with a polypeptide represented by the amino acid sequence of SEQ ID NO: 24-36 (lanes 4-16, respectively), as compared to CD8-positive T cells stimulated with full-length SCD1 protein (lane 3) shows a remarkably high cytotoxic activity against SW480 cells, and ZR75-1 cells. On the other hand, CD8-positive T cells induced using the polypeptide of the negative control is the same level as Mock (lane 1) showed no cytotoxic activity (lane 2). Accordingly, SEQ ID NO: 24-36 are intended to be presented on HLA-A24 molecules on cells expressing human SCD1 protein HLA-A24-positive, this result, the polypeptides of the present invention, like this suggesting that the ability to induce CD8-positive cytotoxic T cells capable of damaging cell.
[0102]
　On the other hand, cancer cells was exposed to the full length SCD1 protein comprising the amino acid sequence represented by the polypeptide and SEQ ID NO: 2 represented by the amino acid sequence of SEQ ID NO: 3-36 with respect to the cancer cell is quite killed There was no. Therefore, these polypeptides was also confirmed that there is no act of killing directly cancer cells.
[0103]
　Cytotoxic activity, as described above, CD8-positive T cells 5 × 10 stimulated induced by each polypeptide used in the present invention 4 10 was incorporated with or chromium 51 3 was mixed with one of each tumor cells the results were cultured for 4 hours, and measuring the amount of chromium 51 released into the medium after culture, showed cytotoxic activity against the tumor cells (referred to target cells) of CD8-positive T cells was calculated by the following equation * Te it is.
[0104]
　* Formula: Cytotoxic activity (%) = CD8-positive T Chromium 51 release amount × 100 cells of chromium 51 release amount ÷ 1N hydrochloric acid target cells were added from the target cells upon addition.
[0105]
　 CD4 for positive T cell antigen epitopes predicted amino acid sequence of human SCD1 protein using computer prediction program SYFPEITHI algorithm (Ramense Author (Rammensee) analyzes, all peptides that selected. select the 9 types of peptides shown in SEQ ID NO: 37-45, which is expected to be HLA class II-binding peptide was synthesized ask custom peptide synthesis services Greiner Japan Co., Ltd. .
[0106]
　Peripheral blood was separated from healthy individuals of the HLA-DRB1 * 04-positive, and centrifuged for 20 minutes at room temperature at 1,500rpm and overlaid on Lymphocyte separation medium (OrganonpTeknika Co., Ltd.). Fractions were collected containing the PBMC, 3 times with cold phosphate buffer (or more) washing to obtain a PBMC. The resulting PBMC were suspended in AIM-V medium 20mL (Life Technololgies Co.), 37 ° C. in a culture flask (manufactured by Falcon), 5% CO 2 and allowed to adhere for 2 hours under the conditions of. Nonadherent cells are used for T cell preparation, adherent cells were used to prepare the dendritic cells.
[0107]
　On the other hand, they were cultured in the presence of IL-4 adherent cells in AIM-V medium (1000 U / mL) and GM-CSF (1000U / mL). IL-4 after 6 days (1000U / mL), GM-CSF (1000U / mL), IL-6 (1000U / mL, manufactured by Genzyme Corporation), IL-1β (10ng / mL, manufactured by Genzyme Corporation), and TNF-alpha was used (10 ng / mL, manufactured by Genzyme Corporation) nonadherent cell population obtained after further cultured for 2 days and replaced with AIM-V medium supplemented with a dendritic cell.
[0108]
　The prepared dendritic cells 1 × 10 in AIM-V medium 6 were suspended at a cell density of cells / mL, the polypeptide of SEQ ID NO: 37-45, in the negative control polypeptide (SEQ ID NO: 48) and SEQ ID NO: 2 the SCD1 protein consisting of the amino acid sequences were added at concentrations of 10mg / mL, 37 ℃ using 96-well plate, 5% CO 2 for 4 hours at conditions. After culturing, and X-ray irradiation (3000 rad), washed with AIM-V medium, (manufactured by Nabi Co.) 10% human AB serum, IL-6 (1000U / mL ) and IL-12 (10ng / mL, Genzyme Corp. ) were suspended in AIM-V medium containing, respectively 1 × 10 per 24-well plate 1 well 5 was added in the cells. Further T cell population, respectively per well 1 × 10 prepared 6 cells were added, 37 ° C., 5% CO 2 and cultured under the conditions of. After 7 days, discarded each culture supernatant (manufactured by Nabi Co.) above and the same way in each peptide and the SCD1 protein obtained after treatment X-ray irradiation dendritic cells with 10% human AB serum and IL-2 (10 U / mL, were suspended in AIM-V medium containing manufactured Genzyme Co.), respectively 1 × 10 per 24-well plate 1 well 5 was added in the cells were further cultured. After repeated 4 times to 7 days every other similar operations, the stimulated T cells were recovered and confirmed the induction of CD4 positive T cells by flow cytometry. As a result, it was confirmed that T cells in each well derived are growing.
[0109]
　
　To examine the specificity for each peptide protein of CD4-positive T cells induced in Example 4 in, and the PBMC that express HLA-DRB1 * 04 molecule in a variety of polypeptides pulsed. The PBMC was added each polypeptide in AIM-V medium at a concentration of 10 [mu] g / mL, 37 ° C., 5% CO 2 was prepared by 4-hour culture under the conditions of. Use In addition, the various polypeptides, each polypeptide represented by the amino acid sequence of SEQ ID NO: 37-45, the full length SCD1 protein comprising the amino acid sequence represented by negative control polypeptide (SEQ ID NO: 48) and SEQ ID NO: 2 It had. 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. Taking supernatant after incubation was measured by ELISA method production of IFN-gamma.
[0110]
　As a result, the culture supernatants of holes with PBMC of each peptide was pulsed in SEQ ID NO: 37 ~ 45, 1000pg / mL or more IFN-gamma has been produced. On the other hand, in the culture supernatant of dendritic cells without the negative control polypeptide or polypeptide pulsed only with (Mock) holes, production of IFN-gamma was hardly observed. Accordingly, various polypeptide represented by the amino acid sequence of SEQ ID NO: 37 through 45 specifically grown stimulate HLA-DRB1 * 04-positive CD4 + T cells, T cell epitope peptide having the ability to induce IFN-gamma production it has been found is. Note that in the amino acid sequence of the full-length SCD1 protein spite contains SEQ ID NO: 37-45 having the immunity-inducing activity, IFN in supernatants hole in culture using PBMC cells pulsed with the full-length SCD1 protein production of -γ was extremely small. It is believed that this did not show sufficient immunity-inducing activity to include many of suppressing sequence immunity-inducing activity in the amino acid sequence of SCD1 protein.
[0111]
　Next, an epitope HLA-DRB1 * 04-positive T cells the polypeptide of SEQ ID NO: 37-45 having the ability to growth stimulation is presented is a process to natural in antigen-presenting cells from SCD1 protein on HLA-DR It was investigated whether it is. SCD1 protein transiently lysates HEK293 cells expressing (purchased from ATCC) was added to immature dendritic cells were digested, after maturation the dendritic cells, the SEQ ID NO: 37-45 polypeptides , T cells stimulated with negative control polypeptides and SCD1 protein was examined whether stimulated by the dendritic cells. Peripheral blood was separated from healthy individuals of the HLA-DRB1 * 04-positive, and centrifuged for 20 minutes at room temperature at 1,500rpm and overlaid on Lymphocyte separation medium. Harvested interphase containing PBMC, cold phosphate buffer three times (or more) and washed to give PBMC. The resulting PBMC were suspended in AIM-V medium 20 mL, 37 ° C. in a culture flask (Falcon), 5% CO 2 deposited 2 hours under the conditions of, IL-4 adherent cells in AIM-V medium ( cultured for 6 days in the presence of 1000 U / mL) and GM-CSF (1000U / mL) , was prepared immature dendritic cells. The lysate 5 × 10 5 was added to the pieces of immature dendritic cells, IL-4 (1000U / mL ), GM-CSF (1000U / mL), IL-6 (1000U / mL), IL-1β (10ng / mL) and TNF-alpha (10 ng / mL) and cultured for 2 days in AIM-V medium supplemented. Dendritic cells after culture were X-ray irradiated (3000 rad), washed with AIM-V medium, suspended in AIM-V medium containing 10% human AB serum, respectively a 96-well plate per hole 3.3 10 × 4Pieces each was added. These 5 × 10 4 to consecutive nucleotides 37-45 of the polypeptide negative control polypeptides and T cells stimulated with SCD1 proteins were added, 37 ° C., 5% CO 2 for 24 hours at conditions. Taking supernatant after incubation was measured by ELISA method production of IFN-gamma.
[0112]
　As a result, as shown in FIG. 6, T cells of SEQ ID NO: 37-45 of the polypeptide lanes 4-12 stimulated with the can to produce IFN-gamma by stimulation of dendritic cells by adding SCD1 protein all right. Meanwhile, in the lane 1 unstimulated in lanes 2 and polypeptides stimulated with a negative control polypeptide, the production of IFN-gamma was hardly observed. Accordingly, the polypeptide of SEQ ID NO: 37-45 are, SCD1 protein revealed that it is a process in natural in antigen presenting cells is an epitope that is presented on HLA-DR. In lane 3 pulsed with full length SCD1 protein In this experiment, production of IFN-gamma was extremely small. Since the in the amino acid sequence of the full-length SCD1 protein is rich in suppressing SEQ an immunity-inducing activity is believed that did not show sufficient immunity-inducing activity.
Industrial Applicability
[0113]
　Immunity-inducing agent comprising a polypeptide exhibiting an anti-tumor activity against various cancer of the present invention, for treating or preventing cancer or useful for the detection of cancer.
[0114]
　All publications cited herein, patents and patent applications are intended to directly incorporated herein by reference.
The scope of the claims
[Requested item 1]
　The following (a) or (b) is selected from the group of polypeptides according to, and at least one polypeptide having immunity-inducing activity,
(a) 34 ~ 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69 ~ 148 of 178 - 195 of 207 - 242 of 247 ~ 280 of the polypeptide consisting of not less than 7 consecutive amino acids 296 - 332 of the region
or according to (b) above (a) one polypeptide 1 to several amino acids are deleted in the amino acid sequence of substitution, insertion or addition polypeptide, or
　a polynucleotide encoding said any one of the polypeptides comprise at least one in vivo immunity-inducing agent containing expressible recombinant vector, as an active ingredient the polypeptide.
[Requested item 2]
　Polypeptide binds to MHC class I molecules with the immunity-inducing activity, immunity-inducing agent according to claim 1.
[Requested item 3]
　Wherein is any one of the polypeptides is a polypeptide having an immunity-inducing activity selected from the group of polypeptides described in the following (c) ~ (e), the immunity-inducing agent according to claim 2.
(c) SEQ ID NO: 3 to an amino acid sequence represented by 36 polypeptide
; (d) (c) 1 - several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide
polypeptide comprising (e) the polypeptide according to (c) or (d) as a partial sequence
[Requested item 4]
　Polypeptide binds to a MHC class II molecule with the immunity-inducing activity, immunity-inducing agent according to claim 1.
[Requested item 5]
　Wherein is any one of the polypeptides is a polypeptide having an immunity-inducing activity selected from the group of polypeptides described in the following (f) ~ (h), the immunity-inducing agent according to claim 4.
(f) comprising the amino acid sequence shown in SEQ ID NO: 37-45 polypeptide
(g) above (f) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide
polypeptide comprising (h) the polypeptide according to (f) or (g) as a partial sequence
[Requested item 6]
　Used as an active ingredient of cancer therapeutic or prophylactic agent, immunity-inducing agent according to any one of claims 1 to 5.
[Requested item 7]
　Wherein the cancer is a cancer that expresses SCD1 protein, immunity-inducing agent according to claim 6.
[Requested item 8]
　Wherein the cancer is malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, renal cancer, colorectal cancer, stomach cancer, malignant brain tumors, esophageal cancer or lung cancer, the immunity-inducing agent according to claim 6 or 7.
[Requested item 9]
　Further comprising an immune enhancer, immunity-inducing agent according to any one of claims 1-8.
[Requested item 10]
　Isolated antigen presenting cell comprising a complex of polypeptides and MHC molecules having an immunity-inducing activity according to claim 1, 3 or 5.
[Requested item 11]
　Isolated T cells that selectively bind the complex of polypeptides and MHC molecules having an immunity-inducing activity according to claim 1, 3 or 5.
[Requested item 12]
　The following (a) or (b) is selected from the group of polypeptides according to and any one of the polypeptide having immunity-inducing activity.
(a) 34 ~ 50 positions in the amino acid sequence shown in SEQ ID NO: 2, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, successive seven 296-332 positions in the region or more polypeptides having an immunity-inducing activity consisting of the amino acid,
(b) the (a) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide.
[Requested item 13]
　The following (i) ~ (iv): (i)
　the following (a) or (b) is selected from the group of polypeptides according to, and at least one polypeptide having immunity-inducing activity:
　　(a) SEQ ID NO: 2 34-50 of the amino acid sequence shown in, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, poly consisting 296-332 of not less than 7 consecutive amino acids in the region of peptide,
　　(b) the 1 to several amino acids in the amino acid sequence of any one of a polypeptide according to (a) deleted, substituted, inserted or added polypeptide;
　(ii) said any one of comprising at least one polynucleotide encoding a polypeptide, capable of expressing recombinant vectors said polypeptide in vivo;
　(iii) a single comprising a conjugate of the any one of the polypeptides and MHC molecules Antigen presenting cells; and
　(iv) said any one of the polypeptides for the specific isolation T cells,
comprising as an active ingredient one or more selected from the group consisting of, therapeutic or preventive agent for cancer.
[Requested item 14]
　Wherein at least one polypeptide is a polypeptide having an immunity-inducing activity selected from the group of polypeptides described in the following (c) ~ (h), treatment or preventive agent for cancer according to claim 13:
( c) a polypeptide consisting of SEQ ID NO: 3-36 amino acid sequence shown in;
(d) the (c) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide;
(e) the (c) or polypeptide comprising a subsequence of the polypeptide according to (d);
(f) consisting of SEQ ID NO: 37-45 amino acid sequence shown in polypeptide;
(g) the ( 1 to several amino acids in the amino acid sequence of the polypeptide are deleted according to f), substituted, inserted or added polypeptide;
(h) the (f) or partial sequence polypeptide according to (g) a polypeptide comprising a.
[Requested item 15]
　Wherein the cancer is a cancer that expresses SCD1 protein, therapeutic or prophylactic agent for cancer according to claim 13 or 14.
[Requested item 16]
　The following (i) ~ (iv): (i)
　the following (a) or (b) is selected from the group of polypeptides according to, and at least one polypeptide having immunity-inducing activity:
　　(a) SEQ ID NO: 2 34-50 of the amino acid sequence shown in, 69-148 positions, 178-195 positions, 207-242 positions, 247-280 positions, poly consisting 296-332 of not less than 7 consecutive amino acids in the region of peptide,
　　(b) the 1 to several amino acids in the amino acid sequence of any one of a polypeptide according to (a) deleted, substituted, inserted or added polypeptide;
　(ii) said any one of comprising at least one polynucleotide encoding a polypeptide, capable of expressing recombinant vectors said polypeptide in vivo;
　(iii) a single comprising a conjugate of the any one of the polypeptides and MHC molecules Antigen presenting cells; and
　(iv) said any one of the polypeptides for the specific isolation T cells,
one or more selected from the group consisting of, which comprises administering to a subject an animal in need thereof, cancer method of treating or preventing.
[Requested item 17]
　Wherein at least one polypeptide is a polypeptide having an immunity-inducing activity selected from the group of polypeptides described in the following (c) ~ (h), The method of claim 16:
(c) SEQ ID NO: 3 polypeptide consisting of the amino acid sequence shown in ~ 36;
(d) the (c) 1 ~ several amino acids in the amino acid sequence of the polypeptide are deleted according to, substituted, inserted or added polypeptide;
(e ) wherein (c) or (a polypeptide comprising as a partial sequence the polypeptide according to d);
; (f) consisting of SEQ ID NO: 37-45 amino acid sequence shown in polypeptide
according to (g) above (f) 1 to several amino acids in the amino acid sequence of the polypeptide are deleted, substituted, inserted or added polypeptide;
(h) the (f) or a polypeptide comprising a polypeptide according as a partial sequence (g).
[Requested item 18]
　Wherein the cancer is a cancer that expresses SCD1 protein, The method of claim 16 or 17.