Immune inducer

Abstract

The present application aims at finding a novel peptide useful as an effective ingredient of a therapeutic or prophylactic agent for cancer, and providing the use of the peptide as an immunological inducer. An immunological inducer containing (a) a peptide comprising the amino acids shown in SEQ ID NOs: 3 to 45, (b) a peptide in which one to several amino acids are deleted, substituted, inserted or added in the peptide of (a) as an effective ingredient is useful as a therapeutic or prophylactic agent for cancer, etc.

Description

[0001] This application is a divisional application of the patent application filed on March 1, 2017, with application number 201780013955.2 and invention title "Immune Inducer". Technical Field

[0002] This invention relates to novel immune inducers that are useful as effective components of drugs for the treatment or prevention of cancer. Background Technology

[0003] SCD1 (stearoyl-CoA desaturase 1) is a protein in which a double bond is introduced at the C9-C10 position of a saturated fatty acid.

[0004] SCD1 protein has been suggested to be associated with cancer development. For example, Non-Patent Literature 1 and 2 disclose that its expression is elevated in various cancers such as liver cancer, esophageal cancer, and colorectal cancer. If the function of SCD1 is inhibited by siRNA or low-molecular-weight inhibitory compounds, the proliferation of cancer cells is suppressed, apoptosis is induced, and existing tumors shrink.

[0005] On the other hand, Patent Document 1 discloses that the SCD1 protein has immune-inducing activity against cancer cells, and is therefore useful in the treatment and prevention of cancer. However, Patent Document 1 does not disclose information about peptides that can bind to MHC molecules.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: WO2012 / 157736

[0009] Non-patent literature

[0010] Non-patent literature 1: Igal RA. Carcinogenesis. Sep; 31(9):1509-15 (2010)

[0011] Non-patent literature 2: Chen L. Sci. Rep. 6, 19665 (2016) Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] The subject of this invention is to discover new polypeptides useful as effective components of drugs for the treatment or prevention of cancer, and to provide the use of such polypeptides as immune inducers.

[0014] In addition, the subject of this invention is to provide isolated antigen-presenting cells comprising a complex of the said polypeptide and MHC molecules, isolated T cells that selectively bind to the complex of the said polypeptide and MHC molecules, and cancer treatments or preventive agents thereof.

[0015] Methods for solving problems

[0016] The inventors of this application conducted in-depth research and obtained the following insights: the human SCD1 protein, composed of the amino acid sequence shown in Serial No. 2, is specifically expressed in tissues or cells of malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, gastric cancer, malignant brain tumors, esophageal cancer, and lung cancer. Furthermore, it was discovered that certain peptides present in specific regions of the SCD1 protein have the ability to be presented by antigen-presenting cells, thereby activating and proliferating T cells specific to that peptide (immunoinducing activity), and that this immunoinducing activity is useful for the treatment or prevention of cancer. Based on these results, it was found that this peptide can be an effective component of an immunoinducing agent for the treatment and / or prevention of cancer; furthermore, antigen-presenting cells in contact with this peptide, and T cells in contact with these antigen-presenting cells, are also useful for the treatment or prevention of cancer, thus completing this invention.

[0017] That is, the present invention has the following features (1) to (12).

[0018] (1) An immune inducer, as an active ingredient, comprising: at least one polypeptide selected from the polypeptide group described in (a) or (b) below and having immune-inducing activity, or a recombinant vector containing at least one polynucleotide encoding any one of the polypeptides and capable of expressing the polypeptide in vivo.

[0019] (a) A polypeptide consisting of seven or more consecutive amino acids in the regions of positions 34–50, 69–148, 178–195, 207–242, 247–280, and 296–332 of the amino acid sequence shown in Serial No. 2.

[0020] (b) A polypeptide in which one or more amino acids are missing, substituted, inserted or added in the amino acid sequence of any of the polypeptides described in (a).

[0021] (2) According to the immune inducer described in (1), the polypeptide with immune-inducing activity can bind to MHC class I molecules.

[0022] (3) According to the immune inducer described in (2), the polypeptide having immune-inducing activity is any polypeptide selected from the group of polypeptides described in (c) to (e) below.

[0023] (c) A polypeptide consisting of the amino acid sequences shown in sequence numbers 3–36.

[0024] (d) A polypeptide in which one or more amino acids are missing, substituted, inserted, or added in the amino acid sequence of the polypeptide described in (c).

[0025] (e) A polypeptide comprising, as part of the sequence, the polypeptide described in (c) or (d).

[0026] (4) According to the immune inducer described in (1), the polypeptide with immune-inducing activity can bind to MHC class II molecules.

[0027] (5) According to the immune inducer described in (4), the polypeptide having immune-inducing activity is any polypeptide selected from the polypeptide group described in (f) to (h) below.

[0028] (f) A polypeptide consisting of the amino acid sequences shown in sequence numbers 37–45.

[0029] (g) A polypeptide whose amino acid sequence is missing, substituted, inserted, or has added one or more amino acids, as described in (f).

[0030] (h) A polypeptide that contains the polypeptide described in (f) or (g) as a partial sequence.

[0031] (6) The immune inducer according to any one of (1) to (5) is used as an active ingredient in a cancer treatment or prevention drug.

[0032] (7) According to the immune inducer described in (6), the cancer is a cancer expressing SCD1 protein.

[0033] (8) The immune inducer according to (6) or (7) is malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, stomach cancer, malignant brain tumor, esophageal cancer or lung cancer.

[0034] (9) The immune inducer according to any one of (1) to (8) further comprises an immune enhancer.

[0035] (10) Isolated antigen-presenting cells containing a complex of an immunogenic polypeptide and an MHC molecule described in (1), (3) or (5).

[0036] (11) Isolated T cells can selectively bind to complexes of MHC molecules containing immunoinducible peptides described in (1), (3) or (5).

[0037] (12) Any polypeptide selected from the polypeptide group described in (a) or (b) below and having immunoinducing activity.

[0038] (a) A polypeptide with immunoinducible activity composed of seven or more consecutive amino acids in the regions of positions 34–50, 69–148, 178–195, 207–242, 247–280, and 296–332 of the amino acid sequence shown in Serial No. 2.

[0039] (b) A polypeptide in which one or more amino acids are missing, substituted, inserted or added in the amino acid sequence of the polypeptide described in (a).

[0040] (13) A drug for the treatment or prevention of cancer, comprising one or more of the following (i) to (iv) as an active ingredient.

[0041] (i) At least one polypeptide selected from the polypeptide group described in (a) or (b) below and having immunoinducing activity:

[0042] (a) A polypeptide consisting of seven or more consecutive amino acids in the regions of positions 34–50, 69–148, 178–195, 207–242, 247–280, and 296–332 of the amino acid sequence shown in Serial No. 2.

[0043] (b) A polypeptide in which one or more amino acids are missing, substituted, inserted or added in the amino acid sequence of any of the polypeptides described in (a);

[0044] (ii) A recombinant vector containing at least one polynucleotide encoding any of the aforementioned polypeptides and capable of expressing the polypeptide in vivo;

[0045] (iii) Isolated antigen-presenting cells comprising a complex of any of the aforementioned polypeptides and an MHC molecule; and

[0046] (iv) T cells isolated specifically for any of the aforementioned polypeptides.

[0047] (14) In the cancer treatment or prevention drug according to (13), the polypeptide with immune-inducing activity is at least one polypeptide selected from the polypeptide group described in (c) to (h) below:

[0048] (c) A polypeptide consisting of the amino acid sequences shown in sequence numbers 3–36.

[0049] (d) A polypeptide in which one or more amino acids are missing, substituted, inserted, or added in the amino acid sequence of the polypeptide described in (c).

[0050] (e) A polypeptide comprising, as a partial sequence, the polypeptide described in (c) or (d).

[0051] (f) A polypeptide consisting of the amino acid sequences shown in sequence numbers 37–45.

[0052] (g) A polypeptide whose amino acid sequence is missing, substituted, inserted, or has added one or more amino acids, as described in (f).

[0053] (h) A polypeptide that contains the polypeptide described in (f) or (g) as a partial sequence.

[0054] (15) A treatment or preventive medicine for cancer according to (13) or (14), wherein the cancer is a cancer expressing the SCD1 protein.

[0055] (16) Methods of treating or preventing cancer, including administering to an animal in need of treatment or prevention of cancer one or more steps selected from the group consisting of (i) to (iv),

[0056] (i) At least one polypeptide selected from the polypeptide group described in (a) or (b) below and having immunoinducing activity:

[0057] (a) A polypeptide consisting of seven or more consecutive amino acids in the regions of positions 34–50, 69–148, 178–195, 207–242, 247–280, and 296–332 of the amino acid sequence shown in Serial No. 2.

[0058] (b) A polypeptide in which one or more amino acids are missing, substituted, inserted or added in the amino acid sequence of any of the polypeptides described in (a);

[0059] (ii) A recombinant vector containing at least one polynucleotide encoding any of the aforementioned polypeptides and capable of expressing the polypeptide in vivo;

[0060] (iii) Isolated antigen-presenting cells comprising a complex of any of the aforementioned polypeptides and an MHC molecule; and

[0061] (iv) T cells isolated specifically for any of the aforementioned polypeptides.

[0062] (17) According to the method of (16), the polypeptide having immune-inducing activity is at least one polypeptide selected from the group of polypeptides described in (c) to (h) below:

[0063] (c) A polypeptide consisting of the amino acid sequences shown in sequence numbers 3–36.

[0064] (d) A polypeptide in which one or more amino acids are missing, substituted, inserted, or added in the amino acid sequence of the polypeptide described in (c).

[0065] (e) A polypeptide comprising, as a partial sequence, the polypeptide described in (c) or (d).

[0066] (f) A polypeptide consisting of the amino acid sequences shown in sequence numbers 37–45.

[0067] (g) A polypeptide whose amino acid sequence is missing, substituted, inserted, or has added one or more amino acids, as described in (f).

[0068] (h) A polypeptide that contains the polypeptide described in (f) or (g) as a partial sequence.

[0069] (18) The cancer is a cancer expressing the SCD1 protein, according to the method described in (16) or (17).

[0070] This specification contains the disclosure of Japanese Patent Application No. 2016-040364, which forms the basis of the priority claim of this application.

[0071] The effects of the invention

[0072] According to the present invention, a novel immune inducer is provided that is useful as an effective ingredient in cancer treatment or prevention.

[0073] Furthermore, as specifically shown in the embodiments described later, the peptides used in this invention can induce immune cells that kill cancer cells, thereby shrinking or regressing existing cancer. Moreover, the peptides used in this invention can enhance the induction of immune cells that kill cancer cells, causing existing cancer to shrink or regress. Therefore, the peptides of this invention are useful as effective components of cancer treatment and prevention drugs. Attached Figure Description

[0074] Figure 1 This is a diagram showing the expression map of the SCD1 gene in human tumor tissues or cancer cell lines. Reference 1: Shows the expression map of the human SCD1 gene. Reference 2: Shows the expression map of the GAPDH gene, which is a human housekeeping gene.

[0075] Figure 2 This diagram shows the recognition of the complex between the polypeptide and HLA-A0201 by CD8-positive T cells specific to each polypeptide consisting of the amino acid sequences shown in sequences 3-23, and the production of IFN-γ. In the diagram, bands 4-24 on the horizontal axis show the IFN-γ production capacity of HLA-A0201-positive CD8-positive T cells stimulated with dendritic cells containing the polypeptides represented by the amino acid sequences shown in sequences 3-23. Band 1 shows the results of the above treatment without the addition of the polypeptide (Mock), Band 2 shows the results of the above treatment with the addition of the negative control polypeptide (outside the scope of this invention, shown in sequence 46), and Band 3 shows the results of the above treatment with the addition of the full-length SCD1 protein consisting of the amino acid sequence shown in sequence 2.

[0076] Figure 3This diagram shows the recognition of the HLA-A24 complex by CD8-positive T cells specific to each polypeptide consisting of the amino acid sequences shown in sequences 24-36, resulting in the production of IFN-γ. In the diagram, bands 4-16 on the horizontal axis show the IFN-γ production capacity of HLA-A24-positive CD8-positive T cells stimulated with dendritic cells containing the polypeptides represented by amino acid sequences 24-36. Band 1 shows the results of the above treatment without the addition of the polypeptide (Mock), band 2 shows the results of the above treatment with the addition of the negative control peptide (outside the scope of this invention, shown in sequence 47), and band 3 shows the results of the above treatment with the addition of the full-length SCD1 protein consisting of the amino acid sequence shown in sequence 2.

[0077] Figure 4A This is a graph showing the cytotoxic activity of CD8-positive T cells against cancer cells specific to each polypeptide consisting of the amino acid sequences shown in sequences 3–23. In the graph, bands 4–24 on the horizontal axis show the cytotoxic activity of HLA-A0201-positive CD8-positive T cells against U251 cells induced by the polypeptides represented by the amino acid sequences shown in sequences 3–23, respectively. Band 1 shows the cytotoxic activity of CD8-positive T cells induced without the addition of the polypeptide (Mock), band 2 shows the cytotoxic activity of CD8-positive T cells induced using the negative control polypeptide (sequence number 46), and band 3 shows the cytotoxic activity of CD8-positive T cells induced using the full-length SCD1 protein consisting of the amino acid sequence shown in sequence number 2.

[0078] Figure 4B This graph shows the cytotoxic activity of CD8-positive T cells against cancer cells, specifically targeting the peptides composed of the amino acid sequences shown in sequences 3–23. In the graph, bands 4–24 on the horizontal axis show the cytotoxic activity of HLA-A0201-positive CD8-positive T cells against SK-Hep-1 cells induced by the peptides represented by the amino acid sequences shown in sequences 3–23. Band 1 shows the cytotoxic activity of CD8-positive T cells induced without the peptide (Mock), band 2 shows the cytotoxic activity of CD8-positive T cells induced using the negative control peptide (sequence number 46), and band 3 shows the cytotoxic activity of CD8-positive T cells induced using the full-length SCD1 protein composed of the amino acid sequence shown in sequence number 2.

[0079] Figure 5AThis graph shows the cytotoxic activity of CD8-positive T cells against cancer cells, specifically targeting the peptides composed of the amino acid sequences shown in sequences 24–36. Bands 4–16 on the horizontal axis show the cytotoxic activity of HLA-A24-positive CD8-positive T cells stimulated with the peptides represented by the amino acid sequences 24–36 against SW480 cells. Band 1 shows the cytotoxic activity of CD8-positive T cells induced without the peptide (Mock), reference number 2 shows the cytotoxic activity of CD8-positive T cells induced with the negative control peptide (sequence number 47), and band 3 shows the cytotoxic activity of CD8-positive T cells induced with the SCD1 protein composed of the amino acid sequence shown in sequence number 2.

[0080] Figure 5B This graph shows the cytotoxic activity of CD8-positive T cells against cancer cells, specifically targeting peptides composed of the amino acid sequences shown in sequences 24–36. Bands 4–16 on the horizontal axis show the cytotoxic activity of HLA-A24-positive CD8-positive T cells stimulated with the peptides represented by the amino acid sequences 24–36 against ZR-75-1 cells. Band 1 shows the cytotoxic activity of CD8-positive T cells induced without the peptide (Mock), reference number 2 shows the cytotoxic activity of CD8-positive T cells induced with the negative control peptide (sequence number 47), and band 3 shows the cytotoxic activity of CD8-positive T cells induced with the SCD1 protein composed of the amino acid sequence shown in sequence number 2.

[0081] Figure 6 This diagram shows the production of IFN-γ by CD4-positive T cells that specifically recognize the complex of each polypeptide with HLA-DRB1*04, as represented by the amino acid sequences shown in sequences 37-45. Bands 4-12 show the IFN-γ production capacity of HLA-DRB1*04-positive CD4-positive T cells stimulated with dendritic cells containing the polypeptides represented by the amino acid sequences shown in sequences 37-45. Band 1 shows the mock results of the above treatment without the addition of the polypeptide, Band 2 shows the results of the above treatment with the addition of the negative control polypeptide (outside the scope of this invention, represented by sequence number 48), and Band 3 shows the results of the above treatment with the addition of the full-length SCD1 protein represented by the amino acid sequence shown in sequence number 2. Detailed Implementation

[0082] <Polypeptide>

[0083] In this invention, "polypeptide" refers to a molecule formed by multiple amino acids linked by peptide bonds. Not only polypeptide molecules with a large number of amino acids, but also low molecular weight molecules (oligopeptides) with fewer amino acids are included in the polypeptides of this invention.

[0084] The polypeptide constituting the immune inducer of the present invention may be selected from the polypeptide group described in (a) or (b) below, and may be at least one polypeptide having immune induction activity.

[0085] (a) A polypeptide consisting of seven or more consecutive amino acids in the regions of positions 34–50 (17 amino acids), 69–148 (80 amino acids), 178–195 (18 amino acids), 207–242 (36 amino acids), 247–280 (34 amino acids), and 296–332 (37 amino acids) with the initiating methionine as the first position in the human SCD1 protein composed of the amino acid sequence shown in Serial No. 2.

[0086] (b) A polypeptide in which one or more amino acids are missing, substituted, inserted or added in the amino acid sequence of the polypeptide described in (a) above.

[0087] Furthermore, in this invention, "composed of... amino acid sequence" means that the amino acid residues are arranged in such an order. Therefore, for example, "a polypeptide composed of the amino acid sequence shown in Serial No. 2" refers to a polypeptide having the amino acid sequence Met Asp ProAla...(omitted)...Tyr Lys Ser Gly shown in Serial No. 2, and having a size of 359 amino acid residues. Additionally, in this specification, for example, "a polypeptide composed of the amino acid sequence shown in Serial No. 2" is sometimes abbreviated as "the polypeptide of Serial No. 2". The same applies to expressions such as "composed of... base sequence".

[0088] Furthermore, in this invention, "immunoinducible activity" refers to the ability to activate and proliferate T cells that respond to cancer cells expressing SCD1 protein. Specifically, this means that: cytotoxic T cells and / or helper T cells stimulated by SCD1 protein or its partial polypeptides have a higher IFN-γ production capacity than unstimulated control T cells; cytotoxic T cells stimulated by SCD1 protein or its partial polypeptides exhibit higher cytotoxic activity against cancer cells expressing SCD1 protein than unstimulated control T cells; helper T cells stimulated by SCD1 protein or its partial polypeptides enhance the cytotoxic activity of cytotoxic T cells compared to unstimulated control T cells; or cytotoxic T cells or helper T cells stimulated by SCD1 protein or its partial polypeptides proliferate better than unstimulated control T cells.

[0089] Cell proliferation can be confirmed by visual observation, microscopic cell counting, flow cytometry, and the amount of tritium-labeled thymidine incorporated into the cells in the culture medium. Furthermore, the ability to produce IFN-γ can be confirmed using, for example, a known enzyme-linked immunospot (ELISpot) assay. Specifically, as described in the examples below, T cells are first co-cultured with a polypeptide (in this invention, SCD1 protein or a subset thereof) whose immune-inducing activity is to be evaluated and antigen-presenting cells from peripheral blood mononuclear cells (hereinafter referred to as "PBMCs"), thereby bringing the T cells into contact with the antigen-presenting cells presenting the polypeptide to be evaluated. Next, the IFN-γ produced by the T cells is measured using an antibody specific to IFN-γ. This allows for the determination of the number of immune cells in the T cells. The immune-inducing activity can be evaluated from the results of this measurement.

[0090] In addition, the determination of cytotoxic activity can be performed, for example, by co-culturing T cells with the polypeptide whose cytotoxic activity is to be evaluated (micro SCD1 protein or a portion thereof in this invention) and antigen-presenting cells from PBMCs, and then investigating in vitro whether they exhibit the ability to inhibit tumor cell proliferation or kill tumor cells (hereinafter referred to as "cytotoxic activity"). The contact between T cells and antigen-presenting cells, as described later, can be achieved by co-culturing both in a liquid culture medium. The determination of cytotoxic activity can be performed, for example, by the method described in Int. J. Cancer, 58: P317, 1994, referred to as... 51 The Cr release assay is performed using a known method.

[0091] When the induced T cells described above are administered to cancer-bearing organisms, the tumor can shrink or regress through the cytotoxic activity of these T cells. Therefore, this immune-inducing activity can also be evaluated as the ability to inhibit the proliferation of cancer cells or to shrink or eliminate cancerous tissue (tumor) (hereinafter referred to as "anti-tumor activity").

[0092] When using the above-mentioned peptides for the treatment or prevention of cancer, although there are no particular limitations, the evaluation of immune-inducing activity is preferably based on cytotoxic activity or antitumor activity.

[0093] As is known in the art, any polypeptide with about 7 amino acid residues or more can contain an epitope, thereby exhibiting antigenicity and immunogenicity, and possessing immune-inducing activity, and thus can be used as the immune inducer of the present invention.

[0094] Therefore, the polypeptide in (a) above is composed of seven or more, preferably eight, nine, or ten or more, consecutive amino acids within the amino acid sequence shown in Serial No. 2, at positions 34-50, 69-148, 178-195, 207-242, 247-280, and 296-332, and possesses immunoinducing activity. Particularly preferred are polypeptides having the amino acid sequence shown in the amino acid sequence shown in Serial No. 2, at positions 34-50, 69-148, 178-195, 207-242, 247-280, and 296-332.

[0095] As a principle of immune induction through the application of cancer antigen peptides, it is known that: the peptide is taken up into an antigen-presenting cell, then broken down into small fragments by peptidase within the cell, and the fragmented antigen peptides are then presented on the surface of the antigen-presenting cell. Cytotoxic T cells and the like recognize the antigen presented on the cell surface and selectively kill cancer cells that have presented the antigen on the cell surface. Furthermore, it is known that helper T cells recognize the antigen presented on the surface of antigen-presenting cells, promoting the induction of selective killing of cancer cells that have presented the antigen on the cell surface by cytotoxic T cells. The antigen peptide presented on the surface of antigen-presenting cells is relatively small, with approximately 7 to 30 amino acids. Therefore, from the viewpoint of presentation on antigen-presenting cells, as the peptide described in (a) above, approximately 7 to 30 consecutive amino acids from the amino acid sequence shown in sequence number 2 (positions 34-50, 69-148, 178-195, 207-242, 247-280, and 296-332) are preferred. The polypeptide is considered adequate if it consists of approximately 8–30, 9–30, or 9–25 amino acids. These smaller polypeptides are sometimes not ingested into the antigen-presenting cell but are presented directly on the cell surface of the antigen-presenting cell.

[0096] Furthermore, the polypeptides taken up by the antigen-presenting cells are cleaved at random positions by peptidases within the cells, producing various polypeptide fragments. These fragments are presented on the surface of the antigen-presenting cells. Therefore, if a large polypeptide, such as those at positions 34-50, 69-148, 178-195, 207-242, 247-280, and 296-332 of the amino acid sequence shown in Serial No. 2, is applied, then through degradation within the antigen-presenting cells, polypeptide fragments effective for immune induction mediated by the antigen-presenting cells will inevitably be generated. Therefore, larger polypeptides can also be used for immune induction mediated by antigen-presenting cells. For example, the number of amino acids in the polypeptide can be 30 or more, preferably 40 or more, more preferably 50 or more, and even more preferably 100 or more.

[0097] Furthermore, the polypeptide of the present invention can be obtained by checking against a cross-linking medium capable of retrieving epitope peptides composed of 8 to 25, preferably 9 to 24, more preferably 9 to 23 amino acids, using methods such as Bioinformatics & Molecular Analysis Selection (BIMAS) HLA Peptide Binding Predictions (http: / / bimas.dcrt.nih.gov / molbio / hla_bind / index.htmL) and SYFPEⅠTHⅠ, which are capable of being epitope peptides. Specifically, the polypeptide of the present invention is a polypeptide composed of seven or more consecutive amino acids in the regions of positions 34 to 50, 69 to 148, 178 to 195, 207 to 242, 247 to 280, and 296 to 332 in the amino acid sequence shown in Serial No. 2. For example, examples of the polypeptides of the present invention include polypeptides shown in serial numbers 3 to 45, or polypeptides comprising a polypeptide consisting of the amino acid sequences shown in serial numbers 3 to 45 as a partial sequence and having 10 to 30 amino acid residues. Among the polypeptides shown in serial numbers 3 to 45 and the polypeptides comprising a polypeptide consisting of the amino acid sequences shown in serial numbers 3 to 45 as a partial sequence and having 10 to 30 amino acid residues, the immunoinducing activity of the polypeptides shown in serial numbers 3 to 36 is exerted by binding to MHC class I molecules, and the immunoinducing activity of the polypeptides shown in serial numbers 37 to 45 is exerted by binding to MHC class II molecules.

[0098] On the other hand, the polypeptide in (b) above is a polypeptide in which one or more amino acid residues are replaced, deleted, inserted, and / or added in the polypeptide in (a) above, and which has immunoinducing activity. For example, as polypeptides of the present invention, polypeptides in which one or more amino acids are deleted, replaced, inserted, or added in the amino acid sequences shown in sequence numbers 3 to 45 can be listed.

[0099] In this specification, "number" in "several" means an integer from 2 to 10, preferably an integer from 2 to 6, more preferably an integer from 2 to 4, and even more preferably an integer from 2 or 3.

[0100] It is generally believed that changes to one or more amino acids in a polypeptide do not affect the function of the original polypeptide, and sometimes even enhance the desired function of the original polypeptide. In fact, it is known that modified peptides consisting of amino acid sequences altered compared to the original amino acid sequence (i.e., substitution, deletion, addition, and / or insertion) 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 described in (b) above can also exert immunoinducing activity, it can be used in the preparation of the immunoinducer of the present invention.

[0101] Furthermore, the 20 amino acids constituting natural proteins can be grouped into groups with similar properties according to their polarity: neutral amino acids with low-polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). It is known that the properties of the polypeptide remain largely unchanged when substitutions are made within each group. Therefore, when replacing amino acid residues in the polypeptide of (a) of the present invention, it is highly likely that immune-inducing activity can be maintained by substitution within these groups, and this is therefore preferred.

[0102] In addition, the polypeptide in (b) above may also be a polypeptide consisting of seven or more consecutive amino acids in the region of positions 34-50, 69-148, 178-195, 207-242, 247-280, and 296-332 in the amino acid sequence shown in Serial No. 2, for example, any polypeptide consisting of the amino acid sequences shown in Serial Nos. 3-45 has 90% or more, preferably 95% or more, more preferably 98% or more, further preferably 99% or more, or 99.5% or more amino acid sequence identity and has immune-inducing activity.

[0103] In this specification, "identity" of an amino acid sequence (or base sequence) refers to the percentage obtained by arranging two amino acid sequences (or base sequences) so that the amino acid residues (or bases) are as consistent as possible, divided by the total number of amino acid residues (or bases). During this arrangement, gaps are appropriately inserted between one or both of the two sequences being compared, as needed. Such sequence arrangement can be performed using well-known procedures such as BLAST, FASTA, and CLUSTALW. When inserting gaps, the total number of amino acid residues becomes the number of residues counted as one amino acid residue for each gap. Therefore, when the total number of counted amino acid residues differs between the two sequences being compared, the sequence identity (%) is calculated by dividing the number of consistent amino acid residues by the total number of amino acid residues in the longer sequence.

[0104] In cases of use related to cancer treatment or prevention, the peptides of the present invention are preferably presented as complexes with various HLA types on the surface of cells or exosomes. Therefore, the peptides of the present invention are preferably selected as peptides that not only possess immunoinducing activity but also have high binding affinity for various HLA types. Thus, peptides with altered binding affinity can also be modified by substitution, insertion, deletion, and / or addition of amino acid residues. In addition to naturally presented peptides, the regularity of the sequences of peptides presented by binding to various HLA types is known (J Immunol, 1994, 152:3913; Immunogenetics, 1995, 41:178; J Immunol, 1994, 155:4307), and modifications based on such regularity can be introduced into the immunogenic peptides of the present invention. For example, to improve HLA-A24 binding affinity, it is desirable to replace the second amino acid at the N-terminus with leucine or methionine, and / or replace the C-terminal amino acid with valine or leucine. Therefore, peptides obtained by replacing the second amino acid at the N-terminus with leucine or methionine, and / or peptides obtained by replacing the C-terminal amino acid with valine or leucine, are included within the scope of this invention.

[0105] Substitution can be introduced not only into the terminal amino acid position, but also into the position where the peptide's TCR can recognize it. Several studies have confirmed that amino acid substitutes of peptides have the same or better immunoinducing activity as the original peptide, such as CAP1, p53 (264-272), Her-2 / neu (369-377), or gp100 (209-217) (Zaremba et al. 1997, Cancer Res. 57: 4570-4577, TKHoffmann et al. 2002, J Immunol. 168(3): 1338-47, SODionne et al. 2003, Cancer Immunol immunother. 52: 199-206, and SODionne et al. 2004, Cancer Immunology, Immunotherapy, 53: 307-314).

[0106] In addition to the modifications described above, the peptides of the present invention can be linked to other substances as long as the resulting linked peptide retains the necessary immunoinducible activity of the original peptide. Examples of other substances are not limited, but include peptides, lipids, sugars and glycans, acetyl groups, natural and synthetic polymers, etc. Modifications such as glycosylation, side-chain oxidation, or phosphorylation can be included, provided that the modification does not impair the biological activity of the original peptide. These types of modifications can be made to impart additional functions (e.g., targeting and delivery functions) or to stabilize the peptide. For example, techniques for introducing D-amino acids, amino acid mimics, or non-natural amino acids to improve the in vivo stability of peptides are well known in the art, and this concept can also be applied to the peptides of the present invention. The stability of the peptide can be determined by several methods. For example, stability can be tested using peptidases, as well as various biological media such as human plasma and serum (e.g., see Verhoef et al., 1986, Eur J Drug Metab Pharmacokin, 11: 291-302).

[0107] Furthermore, the polypeptides of the present invention can be linked to other peptides via spacers or linkers. Examples of other peptides are not limited, and include epitope peptides from other polypeptides. Alternatively, two or more polypeptides of the present invention can be linked via spacers or linkers. The peptides linked via spacers or linkers can be the same or different from each other. The types of spacers and linkers are not particularly limited, and include those composed of peptides, more preferably those composed of peptides having one or more cleavage sites capable of being cleaved by enzymes such as peptidases, proteases, and proteasomes. Examples of linkers or spacers are not limited, but may include AAY (PMDaftarian et al., J Trans Med, 2007, 5:26), AAA, NKRK (RPMSutmuller et al., J Immunol. 2000, 165:7308-7315), or one to several lysine residues (S.Ota et al., 2002, CanRes. 62:1471-1476, KSKawamura et al., 2002, J Immunol. 168:5709-5715). The present invention envisions polypeptides formed by linking other peptides via spacers or linkers.

[0108] In the case where the polypeptides of the present invention contain cysteine ​​residues, these polypeptides tend to form dimers mediated by disulfide bonds between the SH groups of the cysteine ​​residues. Therefore, polypeptide dimers are also included in the polypeptides of the present invention.

[0109] The peptides of this invention can be prepared using well-known techniques. They can be synthesized using chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (tert-butyloxycarbonyl method). Alternatively, they can be synthesized using conventional methods with various commercially available peptide synthesizers.

[0110] Alternatively, using well-known genetic engineering techniques, a polynucleotide encoding the aforementioned polypeptide can be prepared, inserted into an expression vector, and introduced into a host cell. The target polypeptide can then be produced in the host cell, thus obtaining the target polypeptide. When obtaining the target polypeptide from the host cell, purification or separation is performed to ensure that it does not contain any other natural host cell proteins and their fragments, or any other arbitrary chemical substances.

[0111] The polynucleotide encoding the aforementioned polypeptide can be easily prepared using known genetic engineering techniques or commercially available nucleic acid synthesizers via conventional methods. For example, DNA having the base sequence of Serial Number 1 can be prepared by PCR using a pair of primers designed to amplify the base sequence described in Serial Number 1, using human chromosomal DNA or a cDNA library as a template. The PCR reaction conditions can be appropriately set, for example, using a reaction cycle consisting of 94°C for 30 seconds (denaturation), 55°C for 30 seconds to 1 minute (annealing), and 72°C for 2 minutes (extension) as one cycle, and performing, for example, 30 cycles followed by a reaction at 72°C for 1 minute, etc., but not limited to these. Furthermore, based on the base and amino acid sequence information shown in Serial Number 1, suitable probes or primers can be prepared, and by using them to screen cDNA libraries of humans, etc., the desired DNA can be isolated. The cDNA library is preferably prepared from cells, organs, or tissues expressing the protein of Serial Number 2. The preparation of the aforementioned probes or primers, construction of the cDNA library, screening of the cDNA library, and cloning of the target gene are known to those skilled in the art, and can be performed, for example, according to the methods described in 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 Protocol in Molecular Biology: www.currentprotocols.com, etc. DNA encoding the polypeptide described above (a) can be obtained from the DNA thus acquired. Furthermore, since the codons encoding each amino acid are known, the base sequence of the polynucleotide encoding a specific amino acid sequence can be easily determined. Therefore, since the base sequence of the polynucleotide encoding the polypeptide described above (b) can also be easily determined, such a polynucleotide can also be synthesized using a commercially available nucleic acid synthesizer by conventional methods.

[0112] As the host cell mentioned above, any cell capable of expressing the aforementioned polypeptides can be used. Examples of prokaryotic cells include Escherichia coli, while examples of eukaryotic cells include mammalian cultured cells such as monkey kidney cells (COS1) and Chinese hamster ovary cells (CHO), budding yeast, fission yeast, silkworm cells, and African Xenopus oocytes, but are not limited to these.

[0113] When using prokaryotic cells as host cells, expression vectors with origins of replication, promoters, ribosome binding sites, DNA cloning sites, and terminators capable of replication in prokaryotic cells are used. Examples of expression vectors for *E. coli* include the pUC system, pBluescriptII, pET expression system, and pGEX expression system. DNA encoding the aforementioned polypeptide can be inserted into such an expression vector, and after transforming prokaryotic host cells with this vector, the resulting transformants are cultured, allowing the polypeptide encoded by the aforementioned DNA to be expressed in the prokaryotic host cells. In this case, the polypeptide can also be expressed as a fusion protein with other proteins.

[0114] When using eukaryotic cells as host cells, eukaryotic cell expression vectors with promoters, splice regions, and poly-A addition sites are used as expression vectors. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, and pYES2. Similarly, DNA encoding the aforementioned polypeptides can be inserted into such expression vectors, and after transforming eukaryotic host cells with these vectors, the resulting transformants can be cultured to express the polypeptides encoded by the aforementioned DNA in the eukaryotic host cells. When using pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, and pEGFP-C1 as expression vectors, the aforementioned polypeptides can be expressed as fusion proteins with various tags such as His, FLAG, myc, HA, and GFP.

[0115] The expression vector can be introduced into the host cell using well-known methods such as electroporation, calcium phosphate method, liposome method, and DEAE dextran method.

[0116] To isolate and purify a target peptide from a host cell, known separation techniques can be combined. Examples include, but are not limited to, denaturing agents such as urea, surfactant treatment, sonication, enzymatic digestion, salting out, solvent fractionation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric point electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase chromatography.

[0117] Among the polypeptides obtained by the above methods, as described above, there are also polypeptides in fusion protein form fused with any other protein. Examples include fusion proteins fused with glutathione S-transferase (GST) or His tags. Therefore, such polypeptides in fusion protein form are also included within the scope of this invention. Furthermore, polypeptides expressed in transformed cells are sometimes subjected to various intracellular modifications post-translationally. Such post-translational modified polypeptides are also included within the scope of this invention as long as they possess immunoinducible activity. Examples of such translational modifications include N-terminal methionine removal, N-terminal acetylation, addition of glycans, restrictive degradation caused by intracellular proteases, tetradecanoylation, isopreneation, phosphorylation, etc.

[0118] <Immune inducers>

[0119] By administering the immunoinducible polypeptide of the present invention, or an expression vector containing a gene encoding the polypeptide, to cancer-bearing organisms, existing tumors can regress. Furthermore, by administering the aforementioned immunoinducible polypeptide or a gene encoding the polypeptide to precancerous organisms, tumor development can be prevented. Therefore, the polypeptide of the present invention, or the gene encoding the polypeptide, can serve as an effective component of an immunoinducing agent.

[0120] The terms "tumor" and "cancer" refer to malignant neoplasms and can be used interchangeably. In this context, the cancer that is the target is preferably a cancer expressing the SCD1 protein, and particularly preferably malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, stomach cancer, malignant brain tumors, esophageal cancer, and lung cancer.

[0121] The target animal, as described above, is preferably a mammal, more preferably a mammal including primates, pets, livestock, and racing animals, and even more preferably a human, dog, or cat, with a particular preference for a human.

[0122] The cancer-affected individuals (cancer patients when the individual is human) who are the subjects are preferably cancer-affected individuals expressing the SCD1 protein in their bodies, and more specifically, cancer-affected individuals screened by the cancer detection method described in WO2011 / 027807. Particularly preferred are cancer-affected individuals screened based on the higher expression level of the antibody against the SCD1 protein in a sample obtained from the subject organism compared to the higher expression level of the antibody in a sample obtained from a healthy individual. Examples of samples for screening cancer-affected individuals include bodily fluids, tissues, and cells such as blood, serum, plasma, ascites, and pleural effusion; however, when screening by measuring the expression level of the antibody against the SCD1 protein, serum, plasma, ascites, or pleural effusion are preferred.

[0123] The immune inducer of the present invention can be administered orally or non-orally, but non-oral administration, such as intramuscular, subcutaneous, intravenous, or intra-arterial administration, is preferred. When using the immune inducer for the purpose of treating cancer, it can also be administered to the lymph nodes near the tumor to enhance the anti-cancer effect. The dosage is simply the amount effective for immune induction; for example, when used for the treatment or prevention of cancer, any amount effective for the treatment and / or prevention of cancer is acceptable. The effective dosage for the treatment or prevention of cancer should be appropriately selected based on the size of the tumor, symptoms, weight, and volume of the target animal. When the target animal is a human, the effective daily dosage is typically 0.0001–1000 μg, preferably 0.001–1000 μg, which can be administered once or in several divided doses. It is preferred to administer it several times a day, every few days to several months. As specifically illustrated in the examples described later, the immune inducer of the present invention can cause existing tumors to regress. Therefore, since it can also exert an anti-cancer effect on a small number of cancer cells in the early stages of cancer development, its use before cancer onset or after cancer treatment can prevent cancer development and recurrence. In other words, the immune inducer of this invention is useful for both cancer treatment and prevention, and can be an effective ingredient in cancer treatment or prevention drugs.

[0124] The immune inducer of the present invention contains the aforementioned polypeptide of the present invention as an active ingredient, but it can consist of only a single polypeptide or a combination of multiple polypeptides. By combining multiple polypeptides of the present invention, the immune-inducing activity (induction and activation of cytotoxic T cells) of each polypeptide is enhanced, thereby enabling more effective treatment or prevention of cancer.

[0125] The immune inducer of the present invention can be used in combination with known peptides capable of inducing cytotoxic T cells. By combining the polypeptides of the present invention, the immune-inducing activity (cytotoxic T cell induction and activation) of each polypeptide is enhanced, enabling more effective cancer treatment or prevention. Here, "combination" includes administering the immune inducer of the present invention and the known peptides capable of inducing cytotoxic T cells separately or simultaneously. "Administering separately" means administering the immune inducer of the present invention and the known peptides capable of inducing cytotoxic T cells separately with a time difference. The order of administration is not considered. On the other hand, "administering simultaneously" means administering the immune inducer of the present invention and the known peptides capable of inducing cytotoxic T cells in a pre-mixed manner in an integrated manner, or administering the immune inducer of the present invention and the known peptides capable of inducing cytotoxic T cells separately without a time difference.

[0126] The immune inducer of the present invention can be used in combination with other immune enhancers that can strengthen immune responses in vivo. Other immune enhancers may be included in the immune inducer of the present invention, or may be administered to the patient as another composition in combination with the immune inducer of the present invention.

[0127] As one of the aforementioned "other immune enhancers," adjuvants can be cited as an example. Adjuvants can enhance the immune response by providing a reservoir of antigens (extracellular or intracellular), activating macrophages, and stimulating specific lymphocytes, thereby improving anticancer effects. Therefore, when the immune inducer of the present invention is used as an active ingredient in a cancer treatment or prevention drug, the immune inducer preferably contains an adjuvant in addition to the aforementioned polypeptide of the present invention as an active ingredient. Various adjuvants are known in the art, and any adjuvant can be used. Specific examples of adjuvants include MPL (SmithKline Beecham); an analogue obtained by purifying and acid-hydrolyzing the lipopolysaccharides of Salmonella minnesota Re 595; QS21 (SmithKline Beecham); pure QA-21 saponin purified from Quillja saponaria extract; DQS21 described in PCT application WO 96 / 33739 (SmithKline Beecham); QS-7, QS-17, QS-18, and QS-L1 (So, HS, et al., 1997, Molecules and Cells, 7: 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; vitamin E; Montanide; alum; CpG oligonucleotides (e.g., see Kreig, AM, et al.). (al., 1995, Nature 374: 546-549); polyinosinic-polycytidylic acid (poly I:C) and its derivatives (polyICLC, etc.); and various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol. Freund's incomplete adjuvant, Montanide, polyinosinic-polycytidylic acid (poly I:C) and its derivatives, and CpG oligonucleotides are preferred. The mixing ratio of the above adjuvants to the polypeptide is typically about 1:10 to 10:1, preferably about 1:5 to 5:1, and more preferably about 1:1. However, the adjuvants are not limited to the examples above, and adjuvants other than those mentioned above known in the art may also be used when administering the immune inducer of the present invention (e.g., see Goding, Monoclonal Antibodies: Principles and Practice, 2nd edition, 1986). Methods for preparing mixtures or emulsions of immune inducers and adjuvants are well known to those skilled in the art in the field of immunization.

[0128] In addition to the adjuvants mentioned above, factors that stimulate the immune response of the target can also be used as other immune enhancers. For example, various cytokines with properties that stimulate lymphocytes and antigen-presenting cells can be used as immune enhancers in combination with the immune inducer of the present invention. Such a variety of cytokines that can enhance immune responses are well known to those skilled in the art; examples include, but are not limited to, interleukin-12 (IL-12), GM-CSF, IL-18, interferon-α (IFN-α), interferon-β (IFN-β), interferon-ω (IFN-ω), interferon-γ (IFN-γ), and Flt3 ligand, which exhibit enhanced vaccine protective effects. Such factors can also be used as the aforementioned immune enhancers, and can be included in the immune inducer of the present invention, or administered to the patient as another composition in combination with the immune inducer of the present invention.

[0129] <Cancer treatment or prevention drugs>

[0130] The immune inducer of the present invention can be used as an effective ingredient in cancer treatment or prevention drugs.

[0131] Cancer treatment or prevention drugs can be formulated by appropriately mixing the immune inducer of the present invention with pharmacologically permissible additives such as carriers, diluents, and excipients suitable for various administration methods.

[0132] Formulation methods and applicable additives are well-known in the field of pharmaceutical preparations, and any method and additive can be used. Specific examples of additives include diluents such as physiological buffer solutions; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, and glycine; binding agents such as syrups, gelatin, gum arabic, sorbitol, polyvinyl chloride, and tragali gum; and lubricants such as magnesium stearate, polyethylene glycol, talc, and silica, but not limited to these. Formulation forms include oral dosage forms such as tablets, capsules, granules, powders, and syrups; and non-oral dosage forms such as inhalers, injections, suppositories, and liquids. These formulations can be prepared using conventionally known methods.

[0133] <Antigen-presenting cells>

[0134] Furthermore, by contacting the aforementioned polypeptide with antigen-presenting cells in vitro, the polypeptide can be presented to the antigen-presenting cells. That is, the polypeptides in (a) or (b) above can be used as processing agents for antigen-presenting cells. Here, dendritic cells or B cells possessing MHC class I and II molecules are preferably used as antigen-presenting cells. Various MHC class I and II molecules have been identified and are well known. In humans, MHC molecules are called HLA. Examples of HLA class I molecules include HLA-A, HLA-B, and HLA-C, and more specifically, HLA-A1, HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, and HLA-Cw0602. As HLA class II molecules, examples include HLA-DR, HLA-DQ, and HLA-DP. More specifically, examples include 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, and HLA-DPB1.

[0135] Dendritic cells or B cells containing MHC class I or MHC class II molecules can be prepared from blood and other sources using known methods. By inducing dendritic cells from bone marrow, umbilical cord blood, or patient peripheral blood using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 (or IL-4), the addition of tumor-related peptides to this culture system can induce tumor-specific dendritic cells.

[0136] By administering an effective amount of these dendritic cells, the desired immune response in cancer treatment can be induced. The cells used can be bone marrow from a healthy person, umbilical cord blood, the patient's own bone marrow, peripheral blood, etc., but using the patient's own cells is preferred due to their high safety profile and the potential to avoid serious side effects. Peripheral blood or bone marrow can be any of the following: fresh, cryopreserved, or frozen. Peripheral blood can be cultured as whole blood, or only the leukocyte component can be isolated and cultured, but the latter is preferred from an efficiency standpoint. Furthermore, monocytes can be isolated from the leukocyte component. Additionally, in cases where bone marrow or umbilical cord blood is the source, all cells constituting the bone marrow can be cultured, or monocytes can be isolated and cultured from it. Peripheral blood, its leukocyte component, and bone marrow cells contain monocytes that form dendritic cells, hematopoietic stem cells or immature dendritic cells, CD4-positive cells, etc. The cytokines used can be any cytokines with confirmed safety and physiological activity, regardless of whether they are natural or recombinant, or their production method. However, it is preferable to use standard products that ensure quality for medical use by using the minimum required amount. The concentration of the added cytokines is not particularly limited as long as it is sufficient to induce dendritic cells; generally, a total cytokine concentration of about 10–1000 ng / ml is preferred, more preferably about 20–500 ng / ml. Culture can be performed using well-known culture media commonly used for leukocyte culture. The culture temperature is not particularly limited as long as leukocytes can proliferate; approximately 37°C, the human body temperature, is most preferred. Furthermore, the gaseous environment during culture is not particularly limited as long as leukocytes can proliferate; aeration with 5% CO2 is preferred. In addition, the culture period is not particularly limited as long as the required number of cells can be induced; it is generally between 3 days and 2 weeks. Appropriate instruments can be used for cell isolation and culture, but those with confirmed medical safety and stable and easy operation are preferred. In particular, cell culture devices are not limited to common containers such as culture dishes, flasks and culture bottles, but can also use layered containers, multi-segment containers, roller bottles, spinner bottles, bag culture devices, hollow fiber columns, etc.

[0137] The method for contacting the aforementioned peptide with antigen-presenting cells in vitro can itself be carried out by known methods. For example, it can be achieved by culturing the antigen-presenting cells in a culture medium containing the aforementioned peptide. The peptide concentration in the culture medium is not particularly limited, but is typically around 1–100 μg / ml, preferably around 5–20 μg / ml. The cell density during culture is not particularly limited, but is typically 10-1. 3 ~10 7 Approximately 5 × 10⁶ cells / ml, preferably 5 × 10⁶. 4 ~5×10 6Approximately [number] cells / ml. Culture is preferably performed using conventional methods at 37°C and 5% CO2. Furthermore, the peptide length that antigen-presenting cells can typically present on their surface is a maximum of approximately 30 amino acid residues. Therefore, although not particularly limited, in cases where antigen-presenting cells are in vitro in contact with the peptide, the peptide can be prepared to be less than approximately 30 amino acid residues in length.

[0138] By culturing antigen-presenting cells in the presence of the aforementioned peptide, the peptide is inserted into the MHC molecules of the antigen-presenting cells and presented on the cell surface. Therefore, isolated antigen-presenting cells containing a complex of the peptide and an MHC molecule can be prepared using the aforementioned peptide. Such antigen-presenting cells can present the peptide to T cells in vivo or in vitro, inducing and proliferating peptide-specific cytotoxic T cells or helper T cells.

[0139] By in vitro contacting antigen-presenting cells containing the aforementioned polypeptide and MHC molecule complex with T cells, it is possible to induce and proliferate cytotoxic T cells or helper T cells specific to the polypeptide. This can be achieved by co-culturing the antigen-presenting cells and T cells in a liquid culture medium. For example, this can be done by suspending the antigen-presenting cells in a liquid culture medium, placing the resulting suspension in the wells of a microplate, adding T cells, and culturing. The mixing ratio of antigen-presenting cells to T cells during co-culturing is not particularly limited, but a cell ratio of approximately 1:1 to 1:100 is typical, preferably approximately 1:5 to 1:20. The density of antigen-presenting cells suspended in the liquid culture medium is not particularly limited, but is typically approximately 1 million to 10 million cells / mL, preferably approximately 10,000 to 1 million cells / mL. Co-culturing is preferably carried out using conventional methods at 37°C and 5% CO2. The culture time is not particularly limited, but is typically 2 days to 3 weeks, preferably 4 days to 2 weeks. Co-culture is preferably performed in the presence of one or more interleukins such as IL-2, IL-6, IL-7, and IL-12. In this case, the concentrations of IL-2 and IL-7 are typically around 5–20 U / mL, IL-6 around 500–2000 U / mL, and IL-12 around 5–20 ng / mL, but are not limited to these concentrations. The co-culture process described above can be repeated once or several times by adding fresh antigen-presenting cells. For example, the co-culture supernatant can be discarded, and fresh antigen-presenting cell suspension can be added for further co-culture, which can be repeated once or several times. The conditions for each co-culture can be the same as described above.

[0140] Through the aforementioned co-culture, cytotoxic T cells and helper T cells specific to this peptide were induced and proliferated. Therefore, using the aforementioned peptide, isolated T cells that selectively bind to the complex of this peptide and MHC molecules can be prepared.

[0141] As described in the examples below, the gene encoding the SCD1 protein (SCD1 gene) is specifically expressed in malignant lymphoma tissues, malignant lymphoma cells, breast cancer tissues, breast cancer cells, liver cancer tissues, liver cancer cells, prostate cancer tissues, prostate cancer cells, ovarian cancer tissues, ovarian cancer cells, kidney cancer tissues, kidney cancer cells, colorectal cancer tissues, colorectal cancer cells, gastric cancer tissues, gastric cancer cells, malignant brain tumor tissues, malignant brain tumor cells, esophageal cancer tissues, esophageal cancer cells, lung cancer tissues, and lung cancer cells. Therefore, it is considered that the SCD1 protein is present in significantly greater quantities in these cancer types than in normal cells. A portion of the SCD1 protein present in cancer cells is presented by MHC molecules on the surface of cancer cells. If cytotoxic T cells or helper T cells prepared as described above are administered in vivo, the cytotoxic T cells target a portion of the SCD1 protein to kill cancer cells or enhance the cytotoxic activity of the cytotoxic T cells. Furthermore, antigen-presenting cells that present the aforementioned polypeptide can also induce the proliferation of polypeptide-specific cytotoxic T cells and helper T cells in vivo. Therefore, by administering these antigen-presenting cells into the body, cytotoxic T cells can infect cancer cells, or the cytotoxic activity of cytotoxic T cells can be enhanced. In other words, the cytotoxic T cells, helper T cells, and antigen-presenting cells prepared using the aforementioned polypeptide are also useful as cancer treatment or prevention agents, just like the immune inducer of the present invention.

[0142] When administering the separated antigen-presenting cells or separated T cells to an organism, in order to avoid the immune response in the organism attacking these cells as foreign bodies, it is preferable that these separated cells are antigen-presenting cells or T cells collected from the treated patient and prepared using the polypeptides described in (a) or (b) above.

[0143] For cancer treatment or prevention drugs containing antigen-presenting cells or isolated T-cell antigens as active ingredients, the preferred route of administration is non-oral administration, such as intravenous or intra-arterial administration. Furthermore, the dosage is appropriately selected based on symptoms, administration purpose, etc., typically ranging from 1 to 10 trillion cells, preferably from 1 million to 1 billion cells, and preferably administered once every few days or months. The formulation may be a suspension made by suspending the cells in, for example, physiologically buffered saline, or may be used in combination with other anticancer agents, cytokines, etc. Additionally, one or more additives known in the pharmaceutical industry may be added.

[0144] <Gene Vaccine>

[0145] Furthermore, according to the present invention, by expressing the polynucleotide encoding the polypeptide of (a) or (b) above in vivo in a target animal, immune induction, i.e., induction of antibody production and cytotoxic T cells in vivo, can also be achieved, obtaining the same effect as administering the polypeptide. That is, the immune inducer of the present invention may also contain a recombinant vector as an active ingredient, the recombinant vector containing the polynucleotide encoding the polypeptide of (a) or (b) above and capable of expressing the polypeptide in vivo. As shown in the examples described later, such a recombinant vector capable of expressing an antigenic polypeptide is also referred to as a "gene vaccine".

[0146] The vector used for manufacturing gene vaccines is not particularly limited as long as it can be expressed within the cells of the target animal (preferably mammalian cells). It can be a plasmid vector or a viral vector, and any vector known in the field of gene vaccines can be used. Furthermore, the polynucleotides such as DNA and RNA encoding the aforementioned polypeptides can be easily prepared using conventional methods as described above. Additionally, these polynucleotides can be inserted into the vector using methods known to those skilled in the art.

[0147] The preferred routes of administration for gene vaccines are non-oral routes such as intramuscular, subcutaneous, intravenous, and intra-arterial administration. The dosage can be appropriately selected based on factors such as the type of antigen, typically around 0.1 μg to 100 mg per kg of body weight, preferably around 1 μg to 10 mg.

[0148] As a method of utilizing viral vectors, examples include inserting a polynucleotide encoding the aforementioned polypeptide into RNA viruses or DNA viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, poxviruses, polioviruses, and Sindbis viruses, and then infecting target animals with these viruses. Among these methods, the use of retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, etc., is particularly preferred.

[0149] Other methods include direct intramuscular administration of expression plasmids (DNA vaccine method), liposome method, lipid transfection (Lipofectin) method, microinjection method, calcium phosphate method, electroporation method, etc., with DNA vaccine method and liposome method being particularly preferred.

[0150] In order for the gene encoding the above-mentioned polypeptide used in this invention to actually function as a drug, there are in vivo methods that directly introduce the gene into the body and ex vivo methods that collect certain cells from the target animal, introduce the gene into the cells in vitro, and return the cells to the body, with the in vivo method being more preferred.

[0151] When administered via in vivo, the appropriate route of administration can be chosen based on the disease and symptoms for which treatment is intended. For example, it can be administered intravenously, via artery, subcutaneously, or intramuscularly. When administered in vivo, it can be in the form of a liquid formulation, typically formulated as an injectable preparation containing DNA encoding the aforementioned peptide as an active ingredient, and a conventional support carrier can be added as needed. Furthermore, liposomes or membrane fusion liposomes containing this DNA (such as Sendai virus (HVJ) liposomes) can be in the form of suspensions, cryotropic agents, or centrifugation-concentrated cryotropic agents.

[0152] Furthermore, in this invention, when referring to "the base sequence shown in Serial No. 1," it includes not only the base sequence actually shown in Serial No. 1, but also its complementary sequence. Therefore, when referring to "a polynucleotide having the base sequence shown in Serial No. 1," it includes single-stranded polynucleotides having the base sequence actually shown in Serial No. 1, single-stranded polynucleotides having complementary base sequences, and double-stranded polynucleotides composed of them. When preparing the polynucleotide encoding the polypeptide used in this invention, it is suitable to choose any base sequence, a choice that can be easily made by someone skilled in the art.

[0153] Example

[0154] The present invention will now be described in more detail with reference to embodiments.

[0155] <Example 1: Expression Analysis in Various Tissues>

[0156] (1) SCD1 gene expression analysis in various cancer cell lines

[0157] The amino acid sequence of the human SCD1 protein (Sequence No. 1) was obtained from Gene Bank. The expression of the obtained gene in various human cell lines was studied using RT-PCR (Reverse Transcription-PCR). The reverse transcription reaction was performed as follows: using TRIZOL reagent (manufactured by Life Technologies), 50–100 mg of each tissue and 5–10 × 10⁶ mg of each cell line were injected according to the attached protocol. 6Total RNA was extracted from cells. Using this total RNA, cDNA was synthesized according to the attached protocol using the Superscript First-Strand Synthesis System for RT-PCR (Life Technologies). For cDNA from normal human tissues (brain, hippocampus, testes, colon, placenta), GenePool cDNA (Life Technologies), QUICK-Clone cDNA (Clontech), and Large-Insert cDNA Library (Clontech) were used. PCR reactions were performed using primers specific to the obtained genes (primer sequences are described in sequence numbers 49 and 50) as follows. Specifically, reagents and the accompanying buffer were added to a total volume of 25 μl, consisting of 0.25 μl of the sample prepared via reverse transcription, 2 μM of each of the aforementioned primers, 0.2 mM of each dNTP, and 0.65 U of ExTaq polymerase (manufactured by Takarazuchi Co., Ltd.). The mixture was then subjected to 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute using a Thermal Cycler (BIO RAD Co., Ltd.). For comparative control purposes, specific primers for the housekeeping gene GAPDH (the base sequences of the human GAPDH primers are described in sequence numbers 51 and 52) were also used.

[0158] The result is as follows Figure 1 As shown, the human SCD1 gene is expressed in most cancer cell lines, namely malignant lymphoma, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, gastric cancer, malignant brain tumors, esophageal cancer, and lung cancer.

[0159] (2) Expression of SCD1 protein in human cancer tissue (immunohistochemical staining)

[0160] Immunohistochemical staining was performed on 72 cancer tissue samples using paraffin-embedded arrays of various cancer tissues (manufactured by BIOMAX). After treating the human cancer tissue arrays at 60°C for 3 hours, they were placed in staining bottles filled with xylene, with the xylene being replaced every 5 minutes, repeated 3 times. The same procedure was then performed using ethanol and PBS-T instead of xylene. The human cancer tissue arrays were added to staining bottles filled with 10 mM citrate buffer (pH 6.0) containing 0.05% Tween 20, treated at 125°C for 5 minutes, and then incubated at room temperature for at least 40 minutes. Excess moisture around the sections was wiped away with a Kimwipe, surrounded with DAKOPEN, and an appropriate amount of Peroxidase Block (manufactured by DAKO) was added. After incubating at room temperature for 5 minutes, the sections were placed in staining bottles filled with PBS-T, with the PBS-T being replaced every 5 minutes, repeated 3 times. As a blocking solution, PBS-T solution containing 10% FBS was added, and the sections were incubated at room temperature for 1 hour in a humidifier. Next, a solution prepared by adding a commercially available rabbit polyclonal antibody (Sigma-Aldrich) reacting with SCD1 protein to a concentration of 10 μg / mL using PBS-T solution containing 5% FBS was added and incubated overnight at 4°C. After washing three times for 10 minutes with PBS-T, a suitable amount of Peroxidase Labelled Polymer Conjugated (DAKO) was added, and the solution was incubated at room temperature for 30 minutes. After washing three times for 10 minutes with PBS-T, DAB chromogenic solution (DAKO) was added, and the solution was incubated at room temperature for about 10 minutes. The chromogenic solution was then discarded, and the slide was washed three times for 10 minutes with PBS-T, rinsed with distilled water, and then successively immersed in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 1 minute each, followed by incubation in xylene overnight. The slide was then removed, mounted in Glycergel Mounting Medium (DAKO), and observed.

[0161] As a result, the SCD1 protein was found to be strongly expressed in most of the cancers tested, including malignant lymphomas, breast cancer, liver cancer, prostate cancer, ovarian cancer, kidney cancer, colorectal cancer, stomach cancer, malignant brain tumors, esophageal cancer, and lung cancer.

[0162] <Example 2: Induction of peptide epitope-responsive CD8-positive T cells>

[0163] (1) Prediction of peptide motifs combining HLA-A0201 and HLA-A24

[0164] The amino acid sequence information of the human SCD1 protein, serial number 2, was obtained from GenBank. To predict the binding motifs of HLA-A0201 and HLA-A24, the amino acid sequence of the human SCD1 protein was analyzed using a computer prediction program employing the well-known BIMAS software (available at http: / / bimas.dcrt.nih.gov / molbio / hla_bind / ). Twenty-one peptides, consisting of amino acid sequences 3–23, were selected as expected to bind to the HLA-A0201 molecule, and thirteen peptides, consisting of amino acid sequences 24–36, were selected as expected to bind to the HLA-A24 molecule. All selected peptides were synthesized using custom peptide synthesis services from Grainer Japan Co., Ltd. Furthermore, the quality of the synthesized peptides was ensured through HPLC and mass spectrometry analysis.

[0165] (2) Induction of peptide epitope-responsive CD8-positive T cells

[0166] Peripheral blood was isolated from HLA-A0201-positive healthy individuals and stacked on Lymphocyte separation medium (OrganonpTeknika, Durham, NC). The fractions were centrifuged at 1500 rpm at room temperature for 20 minutes. Fractions containing PBMCs were recovered and washed three times (or more) in cold phosphate buffer to obtain PBMCs. The obtained PBMCs were resuspended in 20 mL of AIM-V medium (Life Technologies) and incubated in a culture flask (Falcon) at 37°C and 5% CO2 for 2 hours to allow them to attach. Non-attached cells were used to prepare T cells, and attached cells were used to prepare dendritic cells.

[0167] Attached cells were cultured in AIM-V medium in the presence of IL-4 (1000 U / mL) and GM-CSF (1000 U / mL). After 6 days, the medium was switched to AIM-V medium supplemented with IL-4 (1000 U / mL), GM-CSF (1000 U / mL), IL-6 (1000 U / mL, Genzyme), IL-1β (10 ng / mL, Genzyme), and TNF-α (10 ng / mL, Genzyme). After further culturing for 2 days, the resulting non-attached cell clusters were used as dendritic cells.

[0168] The prepared dendritic cells were used at a concentration of 1×10⁻⁶. 6Cells were suspended at a density of 10 μg / mL in AIM-V medium, and the peptide selected above (1) that is expected to bind to the HLA-A0201 molecule was added at a concentration of 10 μg / mL. The cells were then cultured in 96-well plates at 37°C and 5% CO2 for 4 hours. After culture, the cells were irradiated with X-rays (3000 rad), washed with AIM-V medium, and resuspended in AIM-V medium containing 10% human AB serum (Nabi), IL-6 (1000 U / mL), and IL-12 (10 ng / mL, Genzyme). 1 × 10⁻⁶ of each peptide was added to each well of a 24-well plate. 5 Each cell. Further, 1×10⁻⁶ cells were added to each well of the prepared T cell group. 6 Cells were cultured at 37°C and 5% CO2. After 7 days, the culture supernatants were discarded, and dendritic cells treated with the peptides obtained in the same manner as above and irradiated with X-rays were resuspended in AIM-V medium containing 10% human AB serum (Nabi), IL-7 (10 U / mL, Genzyme), and IL-2 (10 U / mL, Genzyme) (cell density: 1×10⁻⁶). 5 Cells / mL), add 1×10⁻⁶ cells / mL to each well of a 24-well plate. 5 Cells were cultured further. The same procedure was repeated every 7 days, for a total of 4 times. The stimulated T cells were then recovered, and the induction of CD8-positive T cells was confirmed by flow cytometry.

[0169] In addition, as a negative control, a peptide with a sequence outside the scope of the present invention (Sequence No. 46) and an SCD1 protein composed of the amino acid sequence shown in Sequence No. 2, prepared in Example 3 based on WO2012 / 157736, were used as comparative examples and subjected to the same treatment as described above.

[0170] In addition, for peptides expected to bind to the HLA-A24 molecule, dendritic cells and T cell clusters induced from peripheral blood of HLA-A24-positive healthy individuals were used to attempt the induction of peptide epitope-responsive CD8-positive T cells using the same method as described above. Furthermore, as a negative control, a peptide with a sequence outside the scope of this invention (Sequence No. 47) was used, and the SCD1 protein composed of the amino acid sequence shown in Sequence No. 2 was used as a comparative example and subjected to the same treatment.

[0171] <Example 3: Determination of cytotoxic T cell antigenic epitopes>

[0172] (1) IFN-γ production capacity

[0173] To investigate the specificity of T cells induced in Example 2(2) for epitope peptides and proteins, various peptides were pulsed onto dendritic cells expressing the HLA-A0201 molecule. The dendritic cells were prepared by adding each peptide to AIM-V medium at a concentration of 10 μg / mL and culturing at 37°C and 5% CO2 for 4 hours. The various peptides used included the peptides with amino acid sequences 3-23 of the HLA-A0201 molecule expected to bind, a negative control peptide (sequence number 46), and the SCD1 protein composed of the amino acid sequence shown in sequence number 2. 5 × 10⁶ dendritic cells were pulsed. 4 Add 5×10 3 T cells were cultured in 96-well plates for 24 hours in AIM-V medium containing 10% human AB serum. The supernatant was collected after culture, and the production of IFN-γ was determined by ELISA.

[0174] The result was that, compared with bands 1 and 2 of the dendritic cells using the non-pulsed peptide and the negative control peptide, bands 4-24 of the dendritic cells using the peptide represented by the amino acid sequence of sequence numbers 3-23 confirmed significantly higher IFN-γ production. Figure 2 The results showed that peptides 3-23 are T cell epitope peptides with the ability to specifically stimulate the proliferation of HLA-A0201-positive CD8-positive T cells and induce IFN-γ production. Furthermore, the amount of IFN-γ produced using these peptides was significantly higher than that produced by T cells stimulated by the full-length SCD1 protein (with 3) as shown in sequence 2. That is, peptides 3-23 exhibit significantly high immunogenic activity. In addition, although the amino acid sequence of the full-length SCD1 protein shown in sequence 2 contains the aforementioned immunogenic sequences 3-23, the amount of IFN-γ produced by T cells stimulated by the full-length SCD1 protein of sequence 2 was low. This is likely because the amino acid sequence of the full-length SCD1 protein also contains a large number of sequences that inhibit immunogenic activity, thus not exhibiting sufficient immunogenic activity.

[0175] Furthermore, similarly to the above, in order to investigate the specificity of peptide epitope-responsive CD8 positive T cells induced by the peptide epitope-like peptides represented by amino acid sequences 24-36 in Example 3(2), the production of IFN-γ in dendritic cells expressing HLA-A24 molecules of the peptides 24-36 (with amino acid sequences 4-16), the negative control peptide represented by amino acid sequence 47, and the full-length SCD1 protein represented by amino acid sequence 2 was measured by ELISA according to the above method.

[0176] As a result, compared with band 1 of dendritic cells using the pulsed peptide and band 2 of the negative control peptide, significant IFN-γ production was confirmed in the culture supernatant of bands 4–16 of dendritic cells using the pulsed peptide (sequence numbers 24–36). Figure 3 ).

[0177] These results indicate that the peptides in sequences 24–36 are T cell epitope peptides capable of specifically stimulating the proliferation of HLA-A24-positive CD8-positive T cells and inducing IFN-γ production. Furthermore, it was determined that the amount of IFN-γ produced using these peptides was significantly higher than that produced by T cells stimulated with the full-length SCD1 protein as shown in sequence 2. For the same reasons mentioned above, it is considered that the full-length SCD1 protein does not exhibit sufficient immunomodulatory activity.

[0178] (2) Cytotoxicity evaluation

[0179] The next step is to investigate whether the polypeptide represented by the amino acid sequence of sequence numbers 3 to 23 used in this invention is presented on the HLA-A0201 molecule on HLA-A0201 positive tumor cells expressing human SCD1 protein, and whether CD8 positive T cells stimulated by the polypeptide of this invention are toxic to HLA-A0201 positive tumor cells expressing human SCD1 protein, and whether they are significantly more toxic to tumor cells than CD8 positive T cells stimulated by SCD1 protein.

[0180] Ten cells each of the following cell lines expressing human SCD1 protein were selected: U251 cell line (a malignant brain tumor), THP1 leukemia cell line, SK-Hep-1 liver cancer cell line, MCF7 breast cancer cell line, OVCAR3 ovarian cancer cell line, A498 renal cancer cell line, HCT116 colorectal cancer cell line, AGS gastric cancer cell line, and NCI-H522 lung cancer cell line (purchased from JCRB, RIKEN, and ATCC). 6 Each sample was collected in a 50 mL centrifuge tube, and 100 μCi of chromium 51 was added. The tubes were incubated at 37°C for 2 hours. Then, the samples were washed three times with RPMⅠ medium (manufactured by Kibuko) containing 10% fetal bovine serum (hereinafter referred to as FBS, manufactured by Kibuko). 1 × 10⁻⁶ ppm of this medium was added to each well of a 96-well V-plate. 3 Each of the samples was further supplemented with 5 × 10⁵ units of RPM I medium containing 10% FBS. 4HLA-A0201-positive CD8-positive T cells induced by stimulation with the polypeptides shown in amino acid sequences 3–23, the negative control polypeptide (sequence number 46), and the full-length SCD1 protein shown in amino acid sequence 2 were cultured at 37°C and 5% CO2 for 4 hours. After culture, the amount of chromium-51 in the culture supernatant released from the cytotoxic tumor cells was measured to calculate the cytotoxic activity of the CD8-positive T cells induced by each polypeptide and protein.

[0181] The results showed that HLA-A0201-positive CD8-positive T cells induced by stimulation with the peptides represented by amino acid sequences 3-23 exhibited significant cytotoxic activity against all of the aforementioned cell types. As a representative example, Figure 4A and 4B Results showing cytotoxic activity against U251 and SK-Hep-1 cells are presented separately. CD8-positive T cells stimulated with the peptides represented by sequence numbers 3–23 (bands 4–24, respectively) exhibited significantly higher cytotoxic activity against both U251 and SK-Hep-1 cells compared to CD8-positive T cells stimulated with the full-length SCD1 protein (band 3). Conversely, CD8-positive T cells induced with the negative control peptide (band 2) showed no cytotoxic activity, similar to those induced with the Mock peptide (band 1). These results suggest that the peptides used in this invention (sequence numbers 3–23) are presented to the HLA-A0201 molecule on HLA-A0201-positive tumor cells expressing the human SCD1 peptide, thereby enabling the peptides of this invention to induce CD8-positive cytotoxic T cells capable of infecting such tumor cells. Furthermore, although the full-length SCD1 protein contains sequence numbers 3–23, its cytotoxic activity is significantly weaker than that of CD8-positive T cells stimulated with peptides containing sequence numbers 3–23 (bands 3, 4–24). This is likely because the SCD1 protein contains a large number of sequences that inhibit immunogenicity, and therefore cannot induce T cells with strong cytotoxic activity.

[0182] Similarly, the study investigates whether the peptides of sequence numbers 24-36 are presented on HLA-A24 molecules in HLA-A24-positive tumor cells expressing human SCD1 protein, and whether CD8-positive T cells stimulated by the peptides of this invention can be toxic to HLA-A24-positive tumor cells expressing human SCD1 protein, and whether they are significantly more toxic to tumor cells than CD8-positive T cells stimulated by SCD1 protein.

[0183] Chromium-51 was incorporated into human glioma cell lines KNS-42, SK-Hep1, Caki1, SW480, MKN45, PC3, and ZR75-1 (purchased from JCRB, RIKEN, and ATCC) that were HLA-A24 positive and expressed human SCD1 protein. The amount of chromium-51 released from the culture supernatant of toxic cells was measured when HLA-A24 positive CD8 positive T cells stimulated with the peptides shown in the amino acid sequences of sequence numbers 24–36, the negative control peptide (sequence number 47), and the full-length SCD1 protein.

[0184] The results showed that HLA-A24-positive CD8-positive T cells stimulated by the peptides represented by amino acid sequences 24-36 exhibited a significantly higher level of cytotoxic activity against all cancer cells used, an activity that is usually unexpected. As a representative example, Figure 5A and 5B Results of cytotoxic activity against SW480 and ZR75-1 cells are shown separately. CD8-positive T cells stimulated with the peptides represented by amino acid sequences 24–36 (bands 4–16, respectively) showed significantly higher cytotoxic activity against both SW480 and ZR75-1 cells compared to CD8-positive T cells stimulated with the full-length SCD1 protein (band 3). On the other hand, CD8-positive T cells induced with the peptides used as a negative control showed no cytotoxic activity, similar to those in the Mock (band 1) group (band 2). Therefore, sequences 24–36 were presented on the HLA-A24 molecule on HLA-A24-positive cells expressing human SCD1 protein, demonstrating that the peptides of the present invention have the ability to induce CD8-positive cytotoxic T cells capable of infecting such cells.

[0185] On the other hand, exposing the aforementioned cancer cells to the polypeptides represented by amino acid sequences 3-36 and the full-length SCD1 protein composed of the amino acid sequence represented by sequence number 2 resulted in no cancer cell death. This confirms that these polypeptides do not have a direct killing effect on cancer cells.

[0186] Cytotoxic activity, as described above, is demonstrated by stimulating 5 × 10⁵ CD8-positive T cells with the peptides used in this invention. 4 One of 1×10 with chromium 51 added 3 Each tumor cell was mixed and cultured for 4 hours. The amount of chromium 51 released into the culture medium after culture was measured. The cytotoxic activity of CD8 positive T cells against each tumor cell (called target cells) was calculated using the following formula*.

[0187] *Formula: Cytotoxic activity (%) = Amount of chromium-51 released from target cells upon addition to CD8-positive T cells ÷ Amount of chromium-51 released from target cells after addition of 1N hydrochloric acid × 100

[0188] <Example 4: Induction of SCD1 protein-derived peptide epitope-responsive CD4-positive T cells>

[0189] To predict CD4-positive T-cell antigenic epitopes, the amino acid sequence of the human SCD1 protein was analyzed using the SYFPEⅠTHⅠ algorithm (by Rammensee). Nine peptides, sequence numbers 37–45, were selected as potential HLA class II binding peptides. All selected peptides were synthesized using a custom peptide synthesis service provided by Grainer Japan Co., Ltd.

[0190] Peripheral blood was isolated from HLA-DRB1*04 positive healthy individuals and stacked in Lymphocyte separation medium (OrganonpTeknika). The fractions were centrifuged at 1500 rpm at room temperature for 20 minutes. The fractions containing PBMCs were recovered and washed three times (or more) in cold phosphate buffer to obtain PBMCs. The obtained PBMCs were resuspended in 20 mL of AIM-V medium (Life Technologies) and incubated in a culture flask (Falcon) at 37°C and 5% CO2 for 2 hours to allow them to attach. Non-attached cells were used to prepare T cells, and attached cells were used to prepare dendritic cells.

[0191] On the other hand, attached cells were cultured in AIM-V medium in the presence of IL-4 (1000 U / mL) and GM-CSF (1000 U / mL). After 6 days, the medium was switched to AIM-V medium supplemented with IL-4 (1000 U / mL), GM-CSF (1000 U / mL), IL-6 (1000 U / mL, Genzyme), IL-1β (10 ng / mL, Genzyme), and TNF-α (10 ng / mL, Genzyme). After further 2 days of culture, the resulting non-attached cell clusters were used as dendritic cells.

[0192] The prepared dendritic cells were used at a concentration of 1×10⁻⁶. 6Cells were suspended at a density of 10 mg / mL in AIM-V medium. Peptides 37–45, a negative control peptide (serum 48), and the SCD1 protein (composed of the amino acid sequence shown in sequence 2) were added at a concentration of 10 mg / mL. Cells were cultured in 96-well plates at 37°C and 5% CO2 for 4 hours. After culture, the cells were irradiated with X-rays (3000 rad), washed with AIM-V medium, and resuspended in AIM-V medium containing 10% human AB serum (Nabi), IL-6 (1000 U / mL), and IL-12 (10 ng / mL, Genzyme). 1 × 10⁻⁶ cells were added to each well of a 24-well plate. 5 Each cell. Further, 1×10⁻⁶ cells were added to each well of the prepared T cell group. 6 Cells were cultured at 37°C and 5% CO2. After 7 days, the culture supernatants were discarded. Dendritic cells treated with the peptides and SCD1 protein obtained in the same manner as above and then irradiated with X-rays were resuspended in AIM-V medium containing 10% human AB serum (Nabi) and IL-2 (10 U / mL, Genzyme). 1×10⁻⁶ cells were added to each well of a 24-well plate. 5 Cells were further cultured. This process was repeated every 7 days, for a total of 4 times. The stimulated T cells were then recovered, and the induction of CD4-positive T cells was confirmed by flow cytometry. The results confirmed the proliferation of induced T cells in each well.

[0193] <Example 5: Determination of helper T cell antigenic epitopes derived from SCD1 protein in stimulating HLA-DRB1*04 positive CD4 positive T cells>

[0194] To investigate the specificity of CD4-positive T cells induced in Example 4 above for various peptide proteins, PBMCs expressing HLA-DRB1*04 molecules were prepared by pulse expression of various peptides. The PBMCs were prepared by adding each peptide to AIM-V medium at a concentration of 10 μg / mL and culturing at 37°C and 5% CO2 for 4 hours. In addition, the various peptides used were the peptides shown in amino acid sequences 37–45, the negative control peptide (sequence number 48), and the full-length SCD1 protein composed of the amino acid sequence shown in sequence number 2. 5 × 10⁶ PBMCs were pulsed. 4 Add 5×10 4 CD4-positive T cells were cultured in 96-well plates for 24 hours in AIM-V medium containing 10% human AB serum. The supernatant was collected after culture, and the production of IFN-γ was determined by ELISA.

[0195] As a result, IFN-γ levels exceeding 1000 pg / mL were produced in the culture supernatant of PBMCs that had been pulsed with each peptide, sequence numbers 37–45. On the other hand, virtually no IFN-γ production was observed in the culture supernatant of dendritic cells (Mock) that used only the negative control peptide and the unpulsed peptide. Therefore, it was determined that the various peptides represented by the amino acid sequences 37–45 are T cell epitope peptides capable of specifically stimulating the proliferation of HLA-DRB1*04-positive CD4-positive T cells and inducing IFN-γ production. Furthermore, although the full-length SCD1 protein amino acid sequence contains the aforementioned immunogenic sequences 37–45, the amount of IFN-γ produced in the culture supernatant of PBMCs that had been pulsed with the full-length SCD1 protein was extremely low. This is likely because the SCD1 protein amino acid sequence contains a large number of sequences that inhibit immunogenic activity, thus failing to exhibit sufficient immunogenic activity.

[0196] Next, we investigated whether the peptides with sequences 37–45, which possess the ability to stimulate the proliferation of HLA-DRB1*04 positive T cells, are epitopes presented on HLA-DRB1 by the naturally processed SCD1 protein within antigen-presenting cells. Lysate from HEK293 cells (purchased from ATCC) transiently expressing SCD1 protein was added to immature dendritic cells for digestion. After maturation of the dendritic cells, we investigated whether T cells stimulated by the peptides with sequences 37–45, the negative control peptide, and the SCD1 protein were stimulated by these dendritic cells. Peripheral blood was isolated from healthy individuals who were HLA-DRB1*04 positive, stacked on Lymphocyte separation medium, and centrifuged at 1500 rpm at room temperature for 20 minutes. Fractions containing PBMCs were harvested and washed three times (or more) in cold phosphate buffer to obtain PBMCs. The obtained PBMCs were suspended in 20 mL of AIM-V medium (Life Technologies) and allowed to attach in a Falcon culture flask at 37°C and 5% CO2 for 2 hours. The attached cells were then cultured in AIM-V medium for 6 days in the presence of 1 L-4 (1000 U / mL) and GM-CSF (1000 U / mL) to produce immature dendritic cells. The above lysis buffer was added to 5 × 10⁻⁶ cells / mL. 5Immature dendritic cells were cultured for 2 days in AIM-V medium supplemented with IL-4 (1000 U / mL), GM-CSF (1000 U / mL), IL-6 (1000 U / mL), IL-1β (10 ng / mL), and TNF-α (10 ng / mL). The cultured dendritic cells were then irradiated with X-rays (3000 rad), washed with AIM-V medium, and resuspended in AIM-V medium containing 10% human AB serum. 3.3 × 10⁻⁶ cells were added to each well of a 96-well plate. 4 Add 5 x 10 to it. 4 T cells stimulated with the negative control peptides (serial numbers 37-45) and SCD1 protein were cultured at 37°C and 5% CO2 for 24 hours. The supernatant was collected after culture, and the production of IFN-γ was determined by ELISA.

[0197] The result is as follows Figure 6 As shown, T cells in bands 4-12 stimulated by peptides 37-45 produced IFN-γ upon stimulation by dendritic cells containing SCD1 protein. On the other hand, virtually no IFN-γ was observed in band 2 stimulated by the negative control peptide and in band 1 without peptide stimulation. Therefore, it is clear that peptides 37-45 are epitopes of SCD1 protein naturally processed and presented on HLA-DR within antigen-presenting cells. Furthermore, band 3 of the full-length SCD1 protein was also pulsed in this experiment, resulting in very low IFN-γ production. This is likely because the amino acid sequence of the full-length SCD1 protein contains numerous sequences that inhibit immunogenic activity, thus failing to exhibit sufficient immunogenic activity.

[0198] Industry availability

[0199] The immune inducer of the present invention, which contains polypeptides that exert antitumor activity against various cancers, is useful for the treatment or prevention of cancer, or for the detection of cancer.

[0200] All publications, patents and patent applications referenced in this specification are incorporated herein by reference directly.

Claims

1. An immune inducer, as an active ingredient, contains: At least one polypeptide consisting of any of the amino acid sequences shown in sequence numbers 37-45, capable of binding to MHC class II molecules and possessing immunoinducing activity, or A recombinant vector containing at least one polynucleotide encoding any of the aforementioned polypeptides and capable of expressing the polypeptide in vivo.

2. The immune inducer according to claim 1 further comprises an immune enhancer.

3. Isolated antigen-presenting cells comprising a complex of an immunogenic polypeptide and an MHC molecule as described in claim 1.

4. A polypeptide consisting of any of the amino acid sequences shown in sequence numbers 37 to 45, capable of binding to MHC class II molecules and possessing immunoinducing activity.

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