The name of the invention: the immunity-inducing agent
Technical field
[0001]
　The present invention relates to a novel immunity-inducing agent useful for the treatment of cancer and / or prophylactic agent or the like.
Background technique
[0002]
　Cancer is a disease among all of the causes of death, those treatments that are currently practiced in combination with radiation therapy and chemotherapy mainly surgical therapy. Despite the discovery in recent years of new surgical methods of development and new anti-cancer drugs, with the exception of the part of the cancer, the treatment results of cancer is the current situation is not much improved. Recently, cancer antigens recognized by cytotoxic T cells reactive to cancer advances in molecular biology and cancer immunology, such as genes encoding cancer antigens have been identified, expectations for antigen-specific immunotherapy It is growing.
[0003]
　Mast Cell-Expressed Membrane Protein 1 (MCEMP1) is Type 2 transmembrane protein, mast cell-specific manner have been reported to be expressed on the cell membrane, mast cell differentiation, immune response involved in allergic reactions possibility to have been suggested (non-Patent Document 1). However, having an immunity-inducing activity MCEMP1 protein on cancer cells and thereby no reports that the protein is useful in the treatment or prevention of cancer.
CITATION
Non-Patent Document
[0004]
Non-Patent Document 1: Kang Li. et al. Genomics, 86: 68-75 (2005)
Summary of the Invention
Problems that the Invention is to Solve
[0005]
　An object of the present invention is to find a useful novel polypeptides for the treatment of cancer and / or prophylactic agent is to provide the use of the immunity-inducing agent of the polypeptide.
Means for Solving the Problems
[0006]
　The present inventors have conducted extensive research results, SEREX method using serum of the canine testis-derived cDNA library and leukemia canine patient, a cDNA encoding a protein that binds to antibodies present in serum from cancer-bearing living body acquired, based on the cDNA, SEQ ID NO: 4 dogs having the amino acid sequence shown in Mast Cell-Expressed Membrane Protein 1 was produced polypeptide of (hereinafter referred to as MCEMP1). Also, in human acquired canine gene, cat and mouse homology gene based, person having the amino acid sequence shown in SEQ ID NO: 2, 6, 8, to prepare a cat and mouse MCEMP1 polypeptide. And these MCEMP1 polypeptides found leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma, and that is specifically expressed in perianal adenocarcinoma. Furthermore, it was found by administering these MCEMP1 the living body, can induce immune cells to MCEMP1 in vivo, and that can cause regression of tumors in vivo expressing MCEMP1. Furthermore, a polypeptide or expressible recombinant vector a polynucleotide encoding a fragment thereof MCEMP1 were found to induce an antitumor effect in vivo against cancers expressing MCEMP1.
[0007]
　Furthermore, MCEMP1 polypeptide, are presented by antigen presenting cells, it has the ability to activate and proliferate specific cytotoxic T cells to the polypeptide (also referred to as "immunity-inducing activity".) Accordingly, the polypeptide useful in the treatment and / or prevention of cancer, also and antigen-presenting cells contacted with the polypeptide, T cells contacted with the antigen presenting cells in the treatment and / or prevention of cancer It found to be useful, and have completed the present invention.
[0008]
　Accordingly, the present invention includes the features of the following (1) to (11).
(1) is selected from any of the polypeptides of the following (a) ~ (d), and at least one polypeptide having immunity-inducing activity, or a polynucleotide encoding said polypeptide, and in vivo immunity-inducing agent comprising a recombinant vector capable of expressing the polypeptide as an active ingredient.
(A) SEQ ID NO: 2, 4, 6, or a polypeptide comprising the following amino acid full-length of seven or more consecutive amino acid sequence shown in 8 of the sequence listing
SEQ ID NO: (b), the sequence listing 2,4,6 , or one to several amino acids in the amino acid sequence shown in 8 by deletion, polypeptides consisting of substituted or added in the amino acid sequence
amino acids shown in SEQ ID NO: 2, 4, 6, or 8 (c) the sequence listing a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to SEQ
polypeptide comprising a polypeptide of (d) above (a) ~ (c) as a partial sequence
in the processing agent (2) antigen-presenting cells there, immunity-inducing agent according to (1).
(3) as an active ingredient of the cancer therapeutic and / or prophylactic agent, immunity-inducing agent according to (1).
(4) the cancer is a cancer expressing MCEMP1, immunity-inducing agent according to (3).
(5) the cancer is leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, a mastocytoma or perianal adenocarcinoma, immunity-inducing agent according to (3) or (4).
(6) further comprises an immunopotentiator, the immunity-inducing agent according to any one of the above (1) to (5).
(7) The immunity enhancing agent, Freund's incomplete adjuvant, Montanide, poly IC and a derivative thereof, CpG oligonucleotide, interleukin 12, interleukin 18, interferon alpha, interferon beta, interferon omega, interferon gamma, and Flt3 ligand at least one is, the immunity-inducing agent according to (6) selected from the group consisting of.
(8) an antigen presenting cell-derived polypeptide and the test substance as defined in (1) comprises contacting ex vivo or in vitro, method for producing antigen-presenting cell comprising a complex between said polypeptide and the MHC molecule .
(9) The antigen-presenting cell is a dendritic cell or a B cell harboring an MHC class I molecule, the method according to (8).
(10) (8) or (9) and T cells from antigen-presenting cells and a subject obtained by the method according to be contacted ex vivo or in vitro comprising activating the T cells, the the method for manufacturing a cytotoxic T cells specific to the polypeptide as defined in (1).
(11) below (a) ~ is selected from a polypeptide of (d), and at least one polypeptide having immunity-inducing activity, or a polynucleotide encoding the polypeptide, and the polypeptide in vivo the recombinant vector is capable of expressing comprises administering to a subject an immune induction method.
(A) SEQ ID NO: 2, 4, 6, or a polypeptide comprising the following amino acid full-length of seven or more consecutive amino acid sequence shown in 8 of the sequence listing
SEQ ID NO: (b), the sequence listing 2,4,6 , or in the amino acid sequence shown in 8, polypeptide 1 to several amino acids are deleted, replaced or added in the amino acid sequence
(C) a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 2, 4, 6, or 90% or more sequence identity to the amino acid sequence shown in 8 of the Sequence Listing
(d) above (a) ~ (c) polypeptides comprising any of the polypeptide as a partial sequence
　by the present invention, the treatment of cancer and / or novel immunity-inducing agent useful for such prevention is provided. As specifically shown in Examples below, the administration of polypeptides used in the present invention to a subject can induce an immune cell in vivo, further reduction or regression of cancer which have already occurred it can be. Accordingly, the polypeptide is useful for treating or preventing cancer.
[0009]
　This description includes the disclosure of the priority document of the present application Japanese Patent Application No. 2016-064034.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010]
[Figure 1] of the identified MCEMP1 gene is a diagram showing the expression patterns in canine tumor tissue. Reference number 1; dog MCEMP1 gene, indicating the expression pattern of each tumor tissue dogs shown.
[Figure 2] of the identified MCEMP1 gene, shows the expression pattern in various tissues and human cancer cell lines. Reference number 2; human MCEMP1 gene, indicating the expression pattern of each normal human tissue as shown. Reference numeral 3: the human MCEMP1 gene, indicating the expression pattern of each human cancer cell lines as shown. In the figure, PBMC refer to peripheral blood mononuclear cells.
[Figure 3] of the identified MCEMP1 gene, shows the expression pattern of each cancer cell line in mice. Reference number 4; mouse MCEMP1 gene, indicating the expression pattern of each cancer cell line of mice shown.
DESCRIPTION OF THE INVENTION
[0011]
　A more detailed description of the present invention.
1. Polypeptide
　The polypeptide contained as an active ingredient in the immunity-inducing agent of the present invention include the following of (a) ~ (d) a polypeptide. In the present specification, the "polypeptide" refers to a molecule multiple amino acids are formed by peptide bonds, not only the polypeptide molecule number of amino acids is often configuration, the number of amino acids is less low molecular weight molecules and (oligopeptides), full-length protein are also encompassed.
(A) SEQ ID NO: 2, 4, 6 of the Sequence Listing, or consists of the following amino acid full-length of seven or more consecutive in a polypeptide having the amino acid sequence shown in 8, a polypeptide having an immunity-inducing activity.
(B) 1 ~ several amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing is deleted, substituted or added in the amino acid sequence, and polypeptides having an immunity-inducing activity .
(C) SEQ ID NO: 2, 4, 6 of the Sequence Listing, or consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in 8, and a polypeptide having an immunity-inducing activity
(d) above (a) ~ any of the polypeptide comprises a partial sequence, and a polypeptide having an immunity-inducing activity of (c).
[0012]
　In the present invention, the expression "having the amino acid sequence" means that the amino acid residues are aligned in that order. Thus, for example, a "polypeptide having an amino acid sequence shown in SEQ ID NO: 8", Met as shown in SEQ ID NO: 8, His, Ala, Ser, Ala ·· (snip) ·· Gln, Pro, Ser, Thr with the amino acid sequence, it refers to the size of the polypeptide of 183 amino acid residues. Further, for example, it may be abbreviated to "a polypeptide having the amino acid sequence shown in SEQ ID NO: 8" and "polypeptide of SEQ ID NO: 8". The same applies to the expression "having the nucleotide sequence". In this case, the term "comprising" may be replaced by the phrase "consisting of".
[0013]
　Here, "immunity-inducing activity" means the ability to induce the immune cells secrete cytokines such as interferon in a living body.
[0014]
　Whether the polypeptide has an immunity-inducing activity can be confirmed using, for example, known ELISPOT assay, and the like. Specifically, with the cells such as peripheral blood mononuclear cells from an organism treated with the polypeptide to be evaluated immunity-inducing activity, the cells were co-cultured with the polypeptide, cytokine production amount from the cell by measuring using a specific antibody, it is possible to measure the number of immune cells in the cell, thereby evaluating the immunity-inducing activity.
[0015]
　Further, as described in the Examples below, administration of recombinant polypeptide of the (a) ~ (d) in cancer-bearing living body, can also cause regression of the tumor by its immunity-inducing activity. Therefore, the immunity-inducing activity, the ability to shrink or disappear to inhibit the growth of cancer cells or cancer tissue (tumor) (hereinafter referred to as "antitumor activity") can be evaluated as. Antitumor activity of the polypeptide, for example as specifically described in the Examples below, actually confirm the polypeptide I by examining whether the tumor is administered to a tumor-bearing living body is reduced or the like can do. Also, the polypeptide of the cytotoxic T cells induced by administering to the cancer-bearing living body, to evaluate the antitumor activity of the polypeptide by examining whether shows the cytotoxic activity against tumor it can. Measurement of cytotoxic activity of T cells in vivo, for example, limit the T cells to can be confirmed I'll to examine whether tumor is increased such by administering an antibody to be removed from the body not.
[0016]
　Alternatively, the T cells stimulated with the polypeptide (i.e., the polypeptide T cells contacted with the antigen presenting cells presenting) is to examine whether showing cytotoxic activity against tumor cells in vitro Accordingly, it is also possible to evaluate the antitumor activity of the polypeptide. Contact between T cells and antigen presenting cells can be as described below, both performed by cocultivation in liquid media. Measurement of cytotoxic activity, for example, Int. J. Cancer, 58: p317,1994 listed in 51 can be carried out by known method called Cr release assay. When using the polypeptide for the treatment and / or prophylactic use of cancer is not particularly limited, it is preferable to evaluate the immunity-inducing activity antitumor activity as an index.
[0017]
　The amino acid sequence present invention is shown in SEQ ID NO: 2, 4, 6, or 8 of the Sequence Listing to be disclosed, the SEREX method using a canine testis-derived cDNA library and the cancer-bearing dog serum from cancer-bearing dogs polypeptides and human binds to an antibody specifically existing in serum, cats, and was isolated as a mouse homologous factor is the amino acid sequence of MCEMP1 (see example 1). In human MCEMP1 a human homologous factor of the canine MCEMP1, sequence identity of nucleotide sequence 70%, the amino acid sequence 51%, cat phase in cats MCEMP1 sequence identity is the factor nucleotide sequence 83% at the amino acid sequence 64% There, murine phase sequence identity in mice MCEMP1 a same factor nucleotide sequence 65%, the amino acid sequence 47%.
[0018]
　The polypeptides of (a) is SEQ ID NO: 2, 4, 6, or in a polypeptide having the amino acid sequence shown in 8 consecutive 7 or more, preferably from successive 8, 9 or 10 or more amino acids a polypeptide comprising, those having an immunity-inducing activity. Particularly preferably, the polypeptide has the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8. Incidentally, as known in the art, the antigenicity and immunogenicity if the polypeptide than about 7 amino acid residues can exert. Therefore, if SEQ ID NO: 2, 4, 6, or 7 consecutive amino acid residues of the amino acid sequence of the 8 (7) a polypeptide consisting of the entire amino acid residues (full length) or less amino acids from the above, immunity-inducing activity because may have, it can be used for preparation of the immunity-inducing agent of the present invention.
[0019]
　Further, as a principle of immune induction by administration of a cancer antigen polypeptide, the polypeptide is incorporated into antigen-presenting cells, then become smaller fragments undergo degradation by peptidases in said cell, presented on the surface of said cells is, it was recognized cytotoxic T cells and the like, it is known that will kill cells presenting the antigen selectively. The size of polypeptides displayed on the surface of antigen-presenting cells are relatively small, about 7 to 30 amino acids number. Therefore, it from the viewpoint of presentation on antigen presenting cells, the polypeptide of the (a), SEQ ID NO: 2, 4, 6, or continuous about 7 to 30 in the amino acid sequence shown in 8 is one of the preferred embodiments, and more preferably it is sufficient as long as it consists of 8-30 or 9-30 of about amino acid. Polypeptides of relatively small size, without being incorporated into antigen-presenting cells, the program may be presented on the cell surface on direct antigen presenting cells.
[0020]
　Polypeptides incorporated into the antigen-presenting cell receives the disconnection at random positions by a peptidase in said cell, various polypeptide fragments occurs, these polypeptide fragments are presented on the surface of antigen presenting cells Runode, be administered a large size of the polypeptide as full-length region of SEQ ID NO: 2, 4, 6, or 8, by degradation of the antigen-presenting cells, active polypeptide inducing immunity via the antigen-presenting cells fragments inevitably arise. Therefore, also for immune induction via the antigen-presenting cell, can be preferably used a large polypeptide size, the number of amino acid 30 or more, more preferably 100 or more, more preferably 200 or more, more preferably SEQ ID NO: 2, 4 , 6, or may be a polypeptide of the full length region of 8.
[0021]
　The polypeptide of (c) a small number of (preferably one or several) of the polypeptides of (a) amino acid residue is substituted, deleted and / or inserted polypeptide a is the original sequence at least 90%, preferably 95% or more, more preferably 98% or more, more preferably has 99% or more or 99.5% or more sequence identity, and immunity-inducing activity is a polypeptide having an. In general, the protein antigen, a small number of amino acid residues of the amino acid sequence of the protein is replaced, even if it is deleted or inserted, may have substantially the same antigenicity as the original protein it is well known in the art that. Therefore, since the polypeptide (c) described above may exert an immunity-inducing activity, can be used for preparation of the immunity-inducing agent of the present invention. Also, the polypeptide of (b) above, of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8, one to several amino acid residues are substituted, are deleted and / or inserted it is also preferred that a polypeptide. The term "several" as used herein, an integer of 2 to 10, preferably an integer of 2 to 6, more preferably an integer of 2-4.
[0022]
　The term "sequence identity" of amino acid sequence or nucleotide sequence, amino acid residues (or bases) as many matching manner both the amino acid sequences of two amino acid sequences to be compared (or base sequence) (or base sequence) aligning the one in which the matched amino acid residues (or the number of matched bases) represents the value obtained by dividing a percentage by total number of amino acid residues (or the total bases). During the alignment, if necessary, to insert the appropriate gaps in one or both of the two sequences to be compared. Alignment of such sequences, e.g. BLAST, it can be performed using FASTA, a known algorithm such as CLUSTALW. If the gap is inserted, the total number of amino acid residues is a number of residues by counting one gap as one amino acid residue. In this way, the total number of amino acid residues counted is, when the different between the two sequences to be compared, sequence identity (%) is the total number of amino acid residues in the longer sequence, matching amino acid residues It is calculated by dividing the number.
[0023]
　Note that 20 types of amino acids constituting natural proteins, neutral amino acids with side chains having low polarity (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids (Asn having hydrophilic side chains , those having Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, similar properties as Trp) can be grouped, it is often the nature of the polypeptide as long as substitution between these unchanged known. Therefore, when replacing the amino acid residues in the polypeptides of (a) of the present invention, by replacing within the same group, since the possibility of maintaining the immunity-inducing activity increases, preferably .
[0024]
　Polypeptide of (d) above includes any polypeptide described above (a) ~ (c) as a partial sequence, is a polypeptide having an immunity-inducing activity. That is a polypeptide having (a) or (b) of the one or both ends of the polypeptide be one that is added other amino acids or polypeptides, immunity-inducing activity. Such polypeptides can also be used for preparation of the immunity-inducing agent of the present invention.
[0025]
　The above polypeptide, for example, Fmoc method (fluorenyl methyloxy carbonyl method) can be synthesized according to the chemical synthesis methods such as tBoc method (t-butyloxycarbonyl method). It can also be synthesized by a conventional method using various commercially available peptide synthesizers. Further, the using known genetic engineering techniques, to prepare the polynucleotide encoding the polypeptide, is introduced into the host cell incorporating the polynucleotide into an expression vector to produce the polypeptide in said host cell Accordingly, it is possible to obtain the polypeptide of interest.
[0026]
　The polypeptide encoding polynucleotides, by a conventional method using a known genetic engineering technique or commercially available nucleic acid synthesizer, can be readily prepared. For example, DNA having the nucleotide sequence of SEQ ID NO: 3, PCR is performed using a pair of primers designed to the canine chromosomal DNA or cDNA library was used as a template, can be amplified nucleotide sequence described in SEQ ID NO: 3 it can be prepared by. If DNA having the nucleotide sequence of SEQ ID NO: 1, can be similarly prepared by using the human chromosome DNA or cDNA library as the template. The reaction conditions for PCR can be set appropriately, for example, 30 seconds 94 ° C. (denaturation), 30 seconds to 1 minute at 55 ° C. (annealing), 1 cycle reaction step of one minute at 72 ° C. (elongation) as, after for example 30 cycles, and the like can be mentioned conditions for reacting at 72 ° C. 7 minutes, but is not limited thereto. Further, based on the information of the nucleotide sequence and amino acid sequence shown in SEQ ID NO: 1, 3 of the sequence listing herein, to prepare a suitable probe or primer, cDNA libraries, such as human or dog therewith the by screening, it is possible to isolate the desired DNA. cDNA libraries, cells expressing the protein of SEQ ID NO: 2, 4, it is preferably prepared from an organ or tissue. Preparation of the probe or primer, construction of the cDNA library, screening cDNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Molecular black training Second Edition, - Current Protocols it can be carried out according to the method described in in-molecular Biology, and the like. Thus from the obtained DNA as a, can be obtained a DNA encoding a polypeptide of the (a). Moreover, codons encoding each amino acid because it is known, the base sequence of a polynucleotide encoding a specific amino acid sequence can be easily identified. Therefore, encoding a polypeptide of the above (b) ~ (d)
[0027]
　As the host cells, the long polypeptide and capable of expressing cells may be any one, such as E. coli Examples of prokaryotic cells, monkey kidney cells COS1 Examples of eukaryotic cells, Chinese hamster ovary cells cultured mammalian cells such as CHO, budding yeast, fission yeast, silkworm cells, and the like Xenopus egg cells, but are not limited to.
[0028]
　When using a prokaryotic cell as a host cell, the expression vector replicable origin in prokaryotic cells, a promoter, a ribosome binding site, DNA cloning site, an expression vector having a terminator is used. Expression vectors for E. coli, pUC system, pBluescriptII, pET expression system and pGEX expression system. Incorporating DNA encoding the above polypeptide into such an expression vector, after a prokaryotic host cell transformed with the vector, followed by culturing the obtained transformant, a prokaryotic polypeptide which the DNA encodes it can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.
[0029]
　When using eukaryotic cells as host cells, the expression vector, a promoter, a splicing region, a eukaryotic expression vector having a poly (A) addition site and the like is used. Such expression vectors, pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag (A, B, C), pMSG, pYES2 and the like exemplified it can. Similar to the above, incorporating a DNA encoding the above polypeptide into such an expression vector, after a eukaryotic host cell with the vector and transformed by culturing the obtained transformant, the DNA encodes the by which polypeptides can be expressed in eukaryotic host cells. pIND / V5-His as an expression vector, in the case of using the pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like were added His tag, FLAG tag, myc tag, HA tag, various tags such as GFP fusion as a protein, it is possible to express the polypeptide.
[0030]
　Introduction into the host cell expression vectors, electroporation, calcium phosphate method, liposome method, it is possible to use known methods such as DEAE-dextran method.
[0031]
　In order to isolate and purify a polypeptide of interest from the host cells may be carried out by combining known separation operations. For example treatment with denaturing agents such as urea or a surfactant, ultrasonication, enzymatic digestion, salting-out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, Africa Niti chromatography, but reverse phase chromatography, and the like, without limitation.
[0032]
　The polypeptide obtained by the above method, as described above, also include those in the form of a fusion protein with any other protein. For example, such fusion proteins with glutathione -S- transferase (GST) or His tag can be exemplified. Polypeptide in the form of such fusion proteins are also within the scope of the present invention as a polypeptide of the (d). Further, polypeptides expressed in transformed cells after translation may be subject to various modifications within the cell. Such post-translationally modified polypeptides are also as long as it has an immunity-inducing activity, it is included within the scope of the present invention. Examples of such translational modification, elimination of N-terminal methionine, N-terminal acetylation, glycosylation, limited degradation by an intracellular protease, myristoylation, isoprenylation, and phosphorylation.
[0033]
2. Immunity-inducing agent
　as specifically described in the Examples below, administration of a polypeptide having the above-described immunity-inducing activity in cancer-bearing living body, can cause regression of tumors. Therefore, the immunity-inducing agent of the present invention can be used as a therapeutic and / or prophylactic agent for cancer. Further, a polypeptide having the above-described immunity-inducing activity can be used in the treatment and / or prevention of cancer by inducing immunity.
[0034]
　Here, the term "tumor" and "cancer" refers to a malignant neoplasm, are used interchangeably.
[0035]
　In this case, as the cancer of interest, is not particularly limited as long as cancers expressing MCEMP1, preferably expressing significantly more MCEMP1 than normal cell cancer, in particular leukemia, myelodysplasia forming syndrome, osteosarcoma, thymoma, a mastocytoma or perianal adenocarcinoma, these specific cancers, such as acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukemia-free leukemia, leukocyte hypercholesterolemia leukemia (leukocythemic leukemia), basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, gross leukemia, leader cell leukemia, Schilling leukemia, stem cell leukemia, Ashirochi Sexual leukemia, undifferentiated cell leukemia, hairy cell leukemia, blood cell blasts leukemia (hemoblastic leukemia), blood blasts leukemia (hemocytoblastic leukemia), histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenia leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphotropic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia , monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Negeri leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, refractory anemia (RA), refractory anemia with Tetsumedama (RARS), refractory anemia with increased blasts (RAEB), transfer Period RAEB (RAEB-T), preleukemia and chronic myelomonocytic leukemia (CMML), intraosseous normal type osteosarcoma and subline osteosarcoma (bone in well-differentiated osteosarcoma, round cell osteosarcoma, superficial osteosarcoma , beside bone osteosarcoma, periosteal osteosarcoma or table money storage malignant osteosarcoma), thymoma, mastocytoma, perianal adenoma, although perianal adenocarcinoma are included, without limitation.
[0036]
　Also, the subject of interest (animal) is preferably a mammal, more preferably a primate, pet animal ,, zoo animals in captivity, the livestock, in mammals, including sports animals There, particularly preferably a human, dog or cat.
[0037]
　Route of administration to the living body of the immunity-inducing agent of the present invention may be either oral administration or parenteral administration, but intramuscular, subcutaneous, intravenous, parenteral administration, such as intraarterial administration is preferred. When using the immunity-inducing agent for therapeutic purposes of cancer, to enhance the anti-cancer effect it can also be administered to a regional lymph node in the vicinity of the tumor to be treated. The dosage may be an amount effective to induce immunity, for example if used in the treatment and / or prevention of cancer, may be an amount effective for the treatment and / or prevention of cancer. A therapeutically effective amount and / or prophylaxis of cancer is appropriately selected depending on the tumor size and symptoms such as, usually, 0.0001 ~ 1000 [mu] g as an effective amount of one day to the subject animal, preferably 0. is 001 ~ 1000 [mu] g, it can be administered in single or divided doses. Preferably, several times, administered several days to several months apart. As specifically shown in the Examples below, the immunity-inducing agent of the present invention can cause regression of tumors. Accordingly, since the early development of a small number of cancer cells may exert anticancer effects, the use after cancer development before and cancer treatment, it is possible to prevent the onset or recurrence of cancer. That is, the immunity-inducing agent of the present invention is useful for both the prevention and treatment of cancer.
[0038]
　Immunity-inducing agent of the present invention may be composed of only a polypeptide, suitable for each dosage form, pharmacologically acceptable carriers, diluents, by mixing appropriate additives such as excipients It can also be formulated. Formulation methods and available additives are well known in the art of pharmaceutical formulation, it can be used any method and additives. Specific examples of the additives, diluents such as physiological buffer; sugar, lactose, corn starch, calcium phosphate, sorbitol, excipients such as glycine; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth and the like binding agents such as; magnesium stearate, polyethylene glycol, talc, and lubricants such as silica include, but are not limited to. The formulation can include tablets, capsules, granules, powders, oral preparations such as syrups, inhalants, injections, suppositories, and the like parenteral preparations such as solutions. These preparations can be produced by production method it is generally known.
[0039]
　Immunity-inducing agent of the present invention may be used in combination with an immune enhancer that can enhance the immunological response in vivo. Immunopotentiator may be included in the immunity-inducing agent of the present invention may be administered in combination with the immunity-inducing agent of the present invention as separate compositions to the patient.
[0040]
　As the immunopotentiating agent can be, for example, an adjuvant. Adjuvants provides reservoir of antigen (extracellularly or within macrophages), activating macrophages and by stimulating specific sets of lymphocytes, so may enhance the immunological response to enhance the anticancer activity be able to. Thus, in particular, when using the immunity-inducing agent of the present invention for the treatment and / or prevention of cancer, the immunity-inducing agent preferably further comprises an adjuvant in addition to the polypeptide as an active ingredient. Many types of adjuvants are well known in the art, it can be used in any adjuvant. Examples of adjuvants include, MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide purification and congeners obtained after acid hydrolysis of Salmonella; QS21 (SmithKline Beecham), pure purified from Quillja saponaria extract QA-21 saponin; PCT application WO96 / 33739 (SmithKline Beecham) has been described in DQS21; QS-7, QS-17, QS-18 and QS-L1 (Seo (So.), the outer 10 persons, "Morekyuruzu and cell (Molecules and cells) ", 1997, Vol. 7, p.178-186); incomplete Freund's adjuvant; Freund's complete adjuvant; vitamin E; Monta De; alum; CpG oligonucleotide (e.g., Craig (Kreig), the outer seven, "Nature (Nature)", 374 vol, P.546-549) see); poly IC and a derivative thereof (poly ICLC, etc.) as well as various water-in-oil emulsion prepared from squalene and / or biodegradable oils such as tocopherols. Among these, Freund's incomplete adjuvant, Montanide, poly IC and its derivatives, and CpG oligonucleotides are preferred. The mixing ratio of the adjuvant and polypeptide is typically about 1:10 to 10: 1, preferably about 1: 5 to 5 : 1, more preferably from about 1: 1. However, the adjuvant is not limited to the above examples, adjuvants other than known the art may also be used in the administration of the immunity-inducing agent of the present invention (e.g., Goddingu (Goding) al, "Monoclonal Anti Nanobodies: Principles and practice (Monoclonal Antibodies: Principles and practice) ", second edition, 1986). Process for the preparation of mixtures or emulsions of a polypeptide and an adjuvant are well known to those skilled in the vaccination.
[0041]
　Further, as the immunopotentiating agent, in addition to the adjuvant, it is also possible to use factors that stimulate the immune response of the subject. For example, it may be used in combination with the immunity-inducing agent of the present invention the various cytokines having a property to stimulate lymphocytes and antigen-presenting cells as an immune enhancer. Are known in a number of cytokines skilled person capable enhance such immunological response, examples, interleukin-12, which can enhance the protective effects of the vaccine are shown (IL-12), GM-CSF, IL-18, interferon alpha, interferon beta, interferon omega, including but interferon γ and Flt3 ligand, but are not limited to. Such factors can also be used as the immunopotentiating agent and can be as be included in the immunity-inducing agent or a separate composition of the present invention in combination with the immunity-inducing agent of the present invention is administered to a patient.
[0042]
3. Antigen presenting cells or cytotoxic T cells
　present invention further comprises contacting the antigen-presenting cells from above polypeptides and subject ex vivo or in vitro (in vitro), and the polypeptide and the MHC molecule It provides methods of making antigen-presenting cells containing the complex.
[0043]
　The present invention is also obtained by this method, and characterized in that it comprises a complex between the polypeptide and the MHC molecule, to provide antigen-presenting cells.
[0044]
　By contacting the said polypeptide with antigen-presenting cells ex vivo or in vitro, the polypeptide can be presented on antigen-presenting cells. Ie, a polypeptide of the (a) ~ (d) may be employed as the processing agent of antigen-presenting cells. In the present specification, the antigen-presenting cells, may be preferably used dendritic cells or B cells bearing MHC class I molecules. Various MHC class I molecules have been identified and are well known. MHC molecules in humans are referred to as HLA. The HLA class I molecules, HLA-A, HLA-B, there may be mentioned HLA-C, more specifically, HLA-A1, HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, such as HLA-Cw0602 and the like.
[0045]
　Dendritic cells or B cells bearing MHC class I molecules can be prepared from peripheral blood by a well-known method. For example, from bone marrow, umbilical cord blood or patient peripheral blood using granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4) induces dendritic cells, tumor-associated peptide on the culture system by adding, it can induce tumor-specific dendritic cells.
[0046]
　The dendritic cells by administering an effective amount, can induce the desired response in the treatment of cancer. Cells used in bone marrow and umbilical cord blood has been provided from healthy people, but it is possible to use a patient's own bone marrow and peripheral blood, etc., if you use the patient's natural autologous cells, high safety, serious side effects can also be expected be avoided. Peripheral blood or bone marrow may be either fresh sample, cold storage sample and cryopreserved sample. Peripheral blood may be cultured whole blood, only white blood cell component may be cultured in isolation, but the latter is preferred and efficient. It may be further separated mononuclear among white blood cell component. Further, in the case of bone marrow or umbilical cord blood origin may be cultured whole cells constituting the bone marrow may be cultured by separating therefrom mononuclear. Peripheral blood and their white blood cell component, the bone marrow cells, which contain dendritic cell origin become mononuclear hematopoietic stem cells or immature dendritic cells and CD4-positive cells and the like. Cytokines used is not particularly limited as long as safety and physiological activity have been confirmed properties, natural or recombinant type, and the like, but not limited for its production method, preferably the quality to be used in medical is ensured the preparation is used in a necessary minimum amount. Concentration of cytokines to be added is not particularly limited as long as the concentration of the dendritic cells is induced, preferably about 10 ~ 1000 ng / mL in a total concentration of normal cytokine, more preferably about 20 ~ 500ng / mL. Culturing can be carried out using well-known media commonly used for culturing leukocytes. While the culture temperature is not particularly limited as long as the proliferation of white blood cells, and most preferably about 37 ° C. which is human body temperature. Although the gaseous environment in the culture is not particularly limited as long as the proliferation of white blood cells, 5% CO 2It is preferable to vent. Further culture period is not particularly limited as long as a period in which the required number of cells are induced, is usually performed between 3 days to 2 weeks. Equipment to be used for cell separation and culture, can be used as appropriate appropriate, confirmed safety for pharmaceutical use, and it is preferable operation is simple and stable. The particular cell culture device, a petri dish, a flask, regardless generally containers such as bottles, the multilayer container and multistage vessel, roller bottle, spinner type bottle, bag type culturing vessel, may also be used hollow fiber column and the like .
[0047]
　The method itself of contacting the said polypeptide with antigen-presenting cells ex vivo or in vitro can be carried out by well known methods. For example, can be carried out by antigen presenting cells, are cultured in a culture medium containing the polypeptide. Peptide concentration in the medium is not particularly limited, usually about 1 ~ 100 [mu] g / ml, preferably about 5 ~ 20μg / ml. Although cell density is not particularly limited in the culture, usually 10 3 ~ 10 7 cells / ml, preferably about 5x10 4 ~ 5x10 6 , about cells / ml. Culture is carried out according to a conventional method, 37 ° C., 5% CO 2 preferably performed in an atmospheric. The length of the peptide antigen-presenting cells can present on the surface is usually up to about 30 amino acid residues. Therefore, although not particularly limited, when contacting the antigen presenting cells with the polypeptide ex vivo or in vitro, it may be prepared in the following length approximately 30 amino acid residues the polypeptide.
[0048]
　By culturing antigen-presenting cells in the presence of the polypeptide, peptide incorporated into MHC molecules of antigen presenting cells and presented on the surface of antigen-presenting cells. Thus, using the above-described polypeptide, comprising the complex between the polypeptide and the MHC molecule, the isolated antigen-presenting cells can be prepared. Such antigen-presenting cells, in vivo or ex vivo or in vitro, the polypeptide is presented to T cells to induce cytotoxic T cells specific to the polypeptide, can be grown .
[0049]
　The present invention further provides a T cell-derived antigen-presenting cells and subject of the is contacted ex vivo or in vitro comprising activating the T cells, specific cytotoxic to the polypeptide T to provide a method for manufacturing a cell.
[0050]
　The present invention also can be obtained by this method, and is characterized in that is specific to the polypeptides described above, provides a cytotoxic T cell.
[0051]
　Are prepared as described above, the polypeptide and the antigen-presenting cell comprising a complex of MHC molecules, by contacting with T cells and ex vivo or in vitro, specific cytotoxic T to the polypeptide induced cells can be grown. This, and the antigen-presenting cells and T cells can be conducted by co-culturing in a liquid medium. For example, antigen-presenting cells suspended in a liquid medium, placed in a container such as wells of a microplate, can be performed by culturing with the addition of T cells to this. The mixing ratio of antigen-presenting cells and T cells upon cocultivation is not restricted, and usually, 1 at a ratio of cell number: 1 to about 1: 100, preferably 1: 5 to 1: about 20. The density of the antigen-presenting cells suspended in the liquid medium is not particularly limited, usually, from 100 to 10,000,000 cells / ml, preferably about 10,000 to 1,000,000 cells / ml. Coculture is carried out according to a conventional method, 37 ° C., 5% CO 2 preferably performed in an atmospheric. Culture time is not particularly limited, usually 2 days to 3 weeks, preferably 4 days to 2 weeks. Moreover, co-culture is, IL-2, IL-6 , it is preferably carried out in one or more of the presence of interleukin such as IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually 5 ~ 20 U / ml or so, the concentration of IL-6 is usually 500 ~ 2000 U / ml about, concentration of IL-12 is usually about 5 ~ 20 ng / ml some, but not limited thereto. The above cocultivation may be repeated once or several times by adding fresh antigen-presenting cells. For example, discarded culture supernatant after coculture, an operation of performing further co-culture was added to a suspension of fresh antigen-presenting cells, it may be repeated once or several times. Conditions for each cocultivation may be the same as described above.
[0052]
　The cocultivation above, is induced cytotoxic T cells specific to the polypeptide, it is grown. Thus, using the polypeptide, binds selectively to a complex of the polypeptide and the MHC molecule, the isolated T cells can be prepared.
[0053]
　As described in the Examples below, MCEMP1 gene, leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, is specifically expressed in mast cell tumors or perianal adenocarcinoma. Thus, in these cancers is believed that MCEMP1 exists significantly more than normal cells. Therefore, a part of MCEMP1 polypeptides present in the cancer cells to be presented to the MHC molecules on the surface of cancer cells, were prepared as described above cytotoxic T cells are administered in vivo, This cytotoxic T cells can be impaired cancer cells as a marker. Furthermore, antigen-presenting cells that present some MCEMP1 polypeptides also induce cytotoxic T cells specific to the polypeptide in vivo, it is possible to grow, the antigen presenting cells live by administering into the body, it can be impaired cancer cells. That is, the cytotoxic T cells and the antigen-presenting cells prepared using the polypeptide are also similar to the immunity-inducing agent of the present invention, it is useful as therapeutic and / or prophylactic agent for cancer.
[0054]
　When administering the isolated antigen-presenting cells or isolated T cells of the the living body, in order to avoid the immune response in vivo to attack these cells as foreign, antigen presentation taken from the patient to be treated cells or T cells, it is preferable that was prepared using the polypeptide of the as above (a) ~ (d).
[0055]
　The route of administration of the therapeutic and / or prophylactic agent for cancer comprising the antigen-presenting cells or isolated T cells as an active ingredient, parenteral administration, such as intravenous or intraarterial administration is preferred. The dose is appropriately selected depending on the symptoms and the purpose of administration, etc., 1 to 10 trillion usually preferably from 100 million to 1 billion, every several days to every several months preferably administered. Preparations, for example, the cells may be such as those suspended in physiological buffered saline, it may be used in combination with other anticancer agents and cytokines like. It is also possible to add a known one or more additives in the pharmaceutical field.
[0056]
4. DNA vaccines
　by expressing a polynucleotide encoding a polypeptide of the (a) ~ (d) of the target animals in the body, can induce antibody production and cytotoxic T cells in the living body, poly the same effect as administering the peptide is obtained. That is, the immunity-inducing agent of the present invention comprises a polynucleotide encoding a polypeptide of the (a) ~ (d), comprise a expressible recombinant vector the polypeptide as an active ingredient in vivo it may be. As shown in Examples below, recombinant vectors capable of expressing such antigen polypeptide is also referred to as a DNA vaccine.
[0057]
　Vector used to produce the DNA vaccines, the target animal cells (preferably mammalian in animal cells) is not particularly limited as long as it is a vector capable of expressing in may be a viral vector in a plasmid vector, known in the art of DNA vaccines You may use any vector. Incidentally, a polynucleotide such as DNA or RNA encoding the polypeptide, as described above, can be easily prepared by a conventional method. Further, incorporation of the polynucleotide into a vector can be carried out using methods well known to those skilled in the art.
[0058]
　The route of administration of the DNA vaccine preferably parenteral routes of administration intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration, etc., the dosage can be appropriately selected depending on the antigen such as the type usually per body weight 1 kg, about 0.1 [mu] g ~ 100 mg by weight of DNA vaccines, and preferably about 1 [mu] g ~ 10 mg.
[0059]
　The method according to viral vectors, for example retroviruses, adenoviruses, (also referred to as "adeno-associated".) Adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, the RNA virus or DNA virus, such as Sindbis virus incorporate polynucleotides encoding the polypeptides, methods for infecting the like which in the target animal. In this, retrovirus, adenovirus, adeno-associated virus, a method of using vaccinia virus are particularly preferred.
[0060]
　Other methods include a method of administering an expression plasmid directly into muscle (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method and the like, in particular DNA vaccine method, liposome the law is preferable.
[0061]
　To act as a medicament in practice a gene encoding the polypeptide used in the present invention, genes in vivo method for introducing into the body directly, and collected certain cells from a subject animal the cells genes in vitro Although there are ex vivo method of returning the cells in the body it is introduced into the (Nikkei Science, April 1994, p20-45, monthly pharmaceutical Affairs, 1994, Vol. 36, No. 1, p.23-48, experimental medicine Extra Edition, 1994, Vol. 12, No. 15, and the like these references), in vivo method is more preferred.
[0062]
　When administered by in vivo methods it can be administered disease therapeutic purposes, via a suitable administration route depending on the condition and the like. For example, intravenous, intraarterial, subcutaneous, can be administered like intramuscularly. When administered by in vivo method, for example, but may take dosage forms such as solution, in general, is the form of injection containing DNA encoding the polypeptide of the present invention as an active ingredient, requires depending on, which conventional carriers may also be added. Also, the liposome or membrane-fused liposomes containing the DNA (Sendai virus (HVJ) - liposome, etc.) in the suspension, frozen drug, may be in the form of liposomal formulation such as centrifugation-concentrated frozen drug.
[0063]
　In the present specification, for example, when said "base sequence shown in SEQ ID NO: 1", other nucleotide sequences which are actually shown in SEQ ID NO: 1, sequences complementary thereto encompass. Thus, for example, in the case where said "polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1" is a single-stranded polynucleotide having the nucleotide sequence actually shown in SEQ ID NO: 1, its complementary nucleotide sequence single stranded polynucleotide having, and double-stranded polynucleotide composed of these are included. When preparing a polynucleotide encoding the polypeptide used in the present invention is the selecting any one of the nucleotide sequences can be appropriately and easily the selected by those skilled in the art.
Example
[0064]
　Hereinafter, more specifically explained on the basis of the present invention embodiment. However, the scope of the present invention shall not be limited in any way.
[Example 1] SEREX Acquisition of Novel Cancer Antigen Protein by Method
　(1) Preparation of cDNA library
　acids from testis of dogs - guanidinium - phenol - Total RNA was extracted by chloroform method (Acid guanidium-Phenol-Chloroform method), the poly-a RNA was purified according to the protocol attached to the kit by using the Oligotex-dT30 mRNA purification kit (Takara Shuzo Co., Ltd.).
[0065]
　CDNA was synthesized phage library using the obtained mRNA (5 [mu] g). The preparation of cDNA phage library using cDNA Synthesis kit, Zap-cDNA Synthesis Kit, a ZAP-cDNA GigapackIII Gold Cloning Kit ( STRATAGENE Co.) to prepare a library in accordance with the protocol attached to the kit. The size of the cDNA phage library prepared in × 10 1 6 was pfu / ml.
[0066]
　(2) Screening of cDNA library with sera
　using a cDNA phage library prepared above, immunoscreening was carried out. Specifically, infected host E. coli cells (XL1-Blue MRF ') such that about 2500 clones should appear on an NZY agarose plate Φ90 × 15mm, 42 ℃, and cultured for 3 to 4 hours, allow the phage to form plaques , IPTG (isopropyl-beta-D-thiogalactoside) penetration is allowed nitrocellulose membrane (Hybond C Extra: GE Healthecare Ltd. Bio-Sciece Co.) plate to induce and express proteins covering 4 hours at 37 ° C. with, the transfer of the protein to the membrane. To suppress non-specific reactions by subsequently the membrane is recovered TBS containing 0.5% non-fat dry milk (10mM Tris-HCl, 150mM NaCl pH7.5) and shaken overnight at 4 ° C. immersed in. This filter was allowed to react for 2-3 hours at room temperature and the 500-fold diluted canine patient serum.
[0067]
　As the above-described canine patient serum, it was used serum collected from canine patients suffering from leukemia. The serum was stored at -80 ℃, and pretreated immediately before use. Pretreatment method of serum, according to the following method. In other words, they were infected with lambda ZAP Express phage to which no foreign gene was inserted into a host E. coli (XL1-BLue MRF '), 37 ℃ on NZY plate medium and incubated overnight. 0.2 M NaHCO containing 0.5M NaCl in the following 3　 buffers of pH8.3 was added to the plate, 15 hours after standing at 4 ° C., the supernatant was collected as an E. coli / phage extract. Thereafter, the collected E. coli / phage extract NHS- was passed through a column (GE Healthecare Bio-Science Co., Ltd.) to immobilize proteins derived from the E. coli phage. This protein-immobilized column to canine patient serum was allowed to flow through and react remove antibodies adsorbed on E. coli and phage from the serum. The serum fraction that passed through the column was diluted 500-fold with TBS containing 0.5% non-fat dry milk, which was used as immunoscreening material.
[0068]
　After the membrane was blotted the thus treated serum and protein washed 4 times with TBS-T (0.05% Tween20 / TBS), 5000 -fold with TBS containing 0.5% non-fat dry milk as a secondary antibody was diluted goat anti-dog IgG: the (goat anti dog IgG-h + L HRP conjugated BETHYL Laboratories , Inc.), reacted for 1 hour at room temperature, followed by detection by the enzyme coloring reaction using the NBT / BCIP reaction solution (Roche Inc.) , colonies positions where a positive coloring reaction was observed were collected from Φ90 × 15mm of NZY agarose plate, SM buffer (100 mM NaCl, 10 mM MgClSO 4 dissolved, 50mM Tris-HCl, 0.01% gelatin pH 7.5) 500 [mu] l It was. In the same manner as described above until the color reaction positive colonies unifies, secondary, repeated tertiary screening, about 10,000 phage clones reactive with IgG in the serum was screened, a single one of the positive clones release was.
[0069]
　(3) sequence identity search for isolated antigen gene
　for providing a single positive clone isolated by the above method to sequence analysis, performed an operation to convert the phage vector to a plasmid vector. Specifically host E. coli (XL1-Blue MRF ') absorbance OD 600 37 After mixing the solution 200μl which was prepared to become 1.0, the purified phage solution 100μl further ExAssist helper phage and (STRATAGENE Corp.) 1 [mu] l after ° C. 15 min in the reaction of LB medium was 2.5 to 3 hours incubation at 3ml added 37 ° C., was incubated for 20 minutes at immediately 70 ° C. water bath, 4 ° C., centrifuged 1000 × g, 15 min carried out, and the supernatant was collected as a phagemid solution. Phagemid host E. coli (SOLR) absorbance OD then 600 and solution 200μl which was prepared to become 1.0, allowed to react for 15 minutes at 37 ° C. After mixing phage solution 10μl purified, 50 [mu] l ampicillin (final concentration 50 [mu] g / ml ) and incubated 37 ° C. overnight plated on LB agar medium containing. Taken single colony of SOLR was transformed, ampicillin after incubation at (final concentration 50 [mu] g / ml) containing LB medium 37 ° C., purified plasmid DNA having an insert of interest using QIAGEN plasmid Miniprep Kit (Qiagen) did.
[0070]
　Purified plasmid using the T7 primer described in T3 primer SEQ ID NO: 10 as set forth in SEQ ID NO: 9, was analyzed insert the full-length sequence by a primer walking method. And obtaining a gene sequence described in SEQ ID NO: 3 The sequence analysis. Using a base sequence and amino acid sequence of this gene, the sequence was identical search against known genes perform sequence identity search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) the results, obtained gene was found to be MCEMP1 gene. In human MCEMP1 a human homologous factor of the canine MCEMP1, sequence identity of nucleotide sequence 70%, the amino acid sequence 51%, the cat MCEMP1, sequence identity of nucleotide sequence 83%, the amino acid sequence 64%, mouse homologous in mice MCEMP1 a factor, sequence identity of nucleotide sequence and 65% were amino acid sequence 47%. The nucleotide sequence SEQ ID NO: 1 of human MCEMP1, in the amino acid sequence SEQ ID NO: 2, the nucleotide sequence SEQ ID NO: 5 cats MCEMP1, the nucleotide sequence SEQ ID NO: 7 mice MCEMP1 the amino acid sequence in SEQ ID NO: 6, SEQ ID NO: the amino acid sequence It is shown in 8.
[0071]
　(4) Gene expression analysis in each tissue
　dog to gene obtained by the above method, the human and mouse various normal tissues, various tumor tissues and RT-PCR expression in various cancer cell lines (Reverse Transcription-PCR) method It was examined by. Reverse transcription was carried out as follows. That is, each tissue 50 ~ 100 mg and each cell line 5 ~ 10 × 10 6Total RNA was extracted in accordance with the attached protocol using a number of cells from the TRIZOL reagent (Life technology Co., Ltd.). The cDNA was synthesized according to the protocol attached by total RNA Superscript First-Strand Synthesis System for RT-PCR using (Life technology Inc.). cDNA of human normal tissues (brain, testis, colon and placenta), (manufactured by Life technology Ltd.) Gene Pool cDNA, using QUICK-Clone cDNA (Clontech) and Large-Insert cDNA Library (Clontech, Inc.). PCR reactions obtained gene-specific primers (dog primers SEQ ID NO: 11 and 12, the human primers SEQ ID NO: 13 and 14, mice primers SEQ ID NO: 15 and 16) were carried out as follows using. That is, the sample 0.25μl prepared by reverse transcription reaction, the above primers each 2 [mu] M, of each dNTP 0.2 mM, the total volume added to the buffer attached to each reagent so that ExTaq polymerase 0.65u (Takara Shuzo Co., Ltd.) and 25 [mu] l, using a Thermal Cycler (BIO manufactured RAD Co.), 94 ° C. -30 seconds, 55 ° C. -30 seconds, was carried out with 30 cycles of 72 ° C. -1 minutes. As a result, as shown in FIG. 1, dogs MCEMP1 gene in dogs tumor tissue mastocytoma, strong expression in perianal adenocarcinoma was observed (Figure 1). In humans MCEMP1 gene expression, showed no expression in most tissues in healthy human tissues, whereas in the human cancer cell leukemia, myelodysplastic syndromes, strong expression in cell lines of osteosarcoma was observed (Fig. 2) . Furthermore, expression of mouse MCEMP1 gene, leukemia, expressed in cell lines thymoma was detected (Figure 3).
[0072]
Cancer antigen of analysis in vivo of Example 2] MCEMP1
　Preparation of recombinant vector expressing mouse MCEMP1 in (1) in vivo
　based on the nucleotide sequence of SEQ ID NO: 7, by the following method, in vivo in to prepare a recombinant vector expressing the mouse MCEMP1. PCR was prepared from the murine leukemia cell line EL4 seen the expression (purchased from ATCC) in Example 1. The cDNA 1 [mu] l, EcoRI and NotI restriction of two kinds of primers having enzyme cleavage sequence (SEQ ID NO: 17 and 18 according to) each 0.4μM, 0.2mM dNTP, 1.25U of PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.) and the total volume added to the buffer attached to each reagent so that the 50 [mu] l, using a Thermal Cycler (BIO manufactured RAD Co.), 98 ° C. -10 seconds, 55 ° C. -15 seconds, the cycle of 72 ° C. -1 min 30 times It was carried out by repeating. The above-described two kinds of primers were those which amplify the region encoding the entire amino acid sequence of SEQ ID NO: 8. After the PCR, the amplified DNA was electrophoresed on a 1% agarose gel to purify a DNA fragment of about 550bp with the QIAquick Gel Extraction Kit (QIAGEN Inc.).
[0073]
　Were ligated purified DNA fragment into a cloning vector pCR-Blunt (Life technology Inc.). This plasmid was recovered after transformation into E. coli, amplified gene fragments were confirmed by sequencing that it matches the sequence of interest. The matching plasmid sequence of interest was treated with EcoRI and NotI restriction enzyme insertion, and purified using QIAquick Gel Extraction Kit, a target gene sequence, the mammalian expression vector pcDNA3.1 treated with EcoRI and NotI restriction enzymes (Invitrogen Corp.) the (below, mouse MCEMP1 / pcDNA3.1). Mouse MCEMP1 protein is produced in mammalian cells by using this vector.
[0074]
　Above prepared plasmid DNA100μg to 50μg of gold particles (manufactured by Bio Rad Co.), spermidine 100 [mu] l (SIGMA Co.), 1M CaCl 2　 was added 100 [mu] l (SIGMA Co.), and allowed to stand in a stirred for 10 minutes at room temperature by vortexing (referred to as hereinafter gold -DNA particles). After centrifugation for 1 minute at 3000 rpm, washed three times with 100% ethanol supernatant discarded (WAKO Co., Ltd.). After stirring thoroughly by vortexing adding 100% ethanol 6ml gold -DNA particles, pouring gold -DNA particles Tefzel Tubing (manufactured by Bio Rad Co.) was precipitated on the wall. Ethanol gold -DNA particles adhered Tefzel Tubing air dried, and cut to a length suitable for gene gun (hereinafter, mouse MCEMP1 / tube).
[0075]
　(2) Antitumor Effect -1 mouse MCEMP1 by DNA vaccination
　Five A / J mice against (7 weeks old, male, Nippon purchased from SLC Inc.), the tube prepared above (mouse MCEMP1 / tube) were fixed gene gun at a pressure of 400psi with pure helium gas, shaved mice intraperitoneally in DNA vaccine a total of 3 times every 7 days, after administered transdermally (plasmid DNA inoculum 2 [mu] g / mouse become) was then transplanted mouse leukemia cell line EL4 cells was observed with expression of MCEMP1 gene in example 1 to evaluate the anti-tumor effect (prevention model). As a control, it was administered five plasmid DNA mouse MCEMP1 gene is not inserted in the prophylaxis model.
[0076]
　Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. The results of this study, the prevention model, after 28 days, the control group and mice MCEMP1 plasmid administration group, respectively, 1153Mm 3 , 480 mm 3 , and the noticeable regression of tumors in mice MCEMP1 plasmid treated group was observed. Observation of the course of survival in the prevention model, whereas the all cases died 59 days after the administration in the control group, the mice MCEMP1 plasmid treated group, 60% of the mice were alive. From the above results, mice MCEMP1 plasmid treated group significant antitumor effect was observed as compared to the control group.
[0077]
　(3) Antitumor effects -2 mouse MCEMP1 by DNA vaccination
　Five A / J mice against (7 weeks old, male, Nippon purchased from SLC Inc.), the tube prepared above (mouse MCEMP1 / tube) were fixed gene gun at a pressure of 400psi with pure helium gas, shaved mice intraperitoneally in DNA vaccine a total of 3 times every 7 days, after administered transdermally (plasmid DNA inoculum 2 [mu] g / mouse become) was then transplanted mouse leukemia cell line EG7 cells seen with expression of MCEMP1 gene in example 1 to evaluate the anti-tumor effect (prevention model). As a control, it was administered five plasmid DNA mouse MCEMP1 gene is not inserted in the prophylaxis model.
[0078]
　Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. The results of this study, the prevention model, after 28 days, the control group and mice MCEMP1 plasmid administration group, respectively, 982Mm 3 , 521 mm 3 , and the noticeable regression of tumors in mice MCEMP1 plasmid treated group was observed. Observation of the course of survival in the prevention model, whereas the all cases died 68 days after the administration in the control group, the mice MCEMP1 plasmid treated group, 60% of the mice were alive. From the above results, mice MCEMP1 plasmid treated group significant antitumor effect was observed as compared to the control group.
[0079]
　(4) Preparation of recombinant vector expressing human MCEMP1 in vivo
　based on the nucleotide sequence of SEQ ID NO: 1, by the following method, to prepare a recombinant vector expressing human MCEMP1 in vivo. PCR was prepared from the human leukemia cell line U937 seen the expression in Example 1 (purchased from ATCC). The cDNA 1 [mu] l, EcoRI and NotI restriction of two kinds of primers having enzyme cleavage sequences each 0.4μM (SEQ ID NO: 19 and described 20), 0.2mM dNTP, 1.25U of PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.) and the total volume added to the buffer attached to each reagent so that the 50 [mu] l, using a Thermal Cycler (BIO manufactured RAD Co.), 98 ° C. -10 seconds, 55 ° C. -15 seconds, the cycle of 72 ° C. -1 min 30 times It was carried out by repeating. The above-described two kinds of primers were those which amplify the region encoding the entire amino acid sequence of SEQ ID NO: 2. After the PCR, the amplified DNA was electrophoresed on a 1% agarose gel to purify a DNA fragment of about 550bp with the QIAquick Gel Extraction Kit (QIAGEN Inc.).
[0080]
　Were ligated purified DNA fragment into a cloning vector pCR-Blunt (Life technology Inc.). This plasmid was recovered after transformation into E. coli, amplified gene fragments were confirmed by sequencing that it matches the sequence of interest. The matching plasmid sequence of interest was treated with EcoRI and NotI restriction enzyme insertion, and purified using QIAquick Gel Extraction Kit, a target gene sequence, the mammalian expression vector pcDNA3.1 treated with EcoRI and NotI restriction enzymes (Invitrogen Corp.) the (hereinafter referred to as human MCEMP1 / pcDNA3.1). Human MCEMP1 protein is produced in mammalian cells by using this vector.
[0081]
　Above prepared plasmid DNA100μg to 50μg of gold particles (manufactured by Bio Rad Co.), spermidine 100 [mu] l (SIGMA Co.), 1M CaCl 2　 was added 100 [mu] l (SIGMA Co.), and allowed to stand in a stirred for 10 minutes at room temperature by vortexing (referred to as hereinafter gold -DNA particles). After centrifugation for 1 minute at 3000 rpm, washed three times with 100% ethanol supernatant discarded (WAKO Co., Ltd.). After stirring thoroughly by vortexing adding 100% ethanol 6ml gold -DNA particles, pouring gold -DNA particles Tefzel Tubing (manufactured by Bio Rad Co.) was precipitated on the wall. Ethanol gold -DNA particles adhered Tefzel Tubing air dried, and cut to a length suitable for gene gun (hereinafter, human MCEMP1 / tube).
[0082]
　(5) Establishment of full-length human MCEMP1 constant expressing cells
　Human MCEMP1 / pcDNA3.1 prepared above were introduced by lipofection into mouse neuroblastoma cell line N2a cells (ATCC Corp.), 500 [mu] g / ml of G418 (Nacalai Inc. the selection by), were established N2a cell line expressing full-length human MCEMP1 constantly (N2A human MCEMP1). Expression vectors cDNA encoding human MCEMP1 is not inserted (hereinafter, emp / pcDNA3.1) was used as a control cell cells were selected by introducing in the same manner as above (hereinafter, N2a-emp).
[0083]
　(6) Anti-tumor effect of human MCEMP1 by DNA vaccination
　Five A / J mice against (7 weeks old, male, Nippon purchased from SLC Inc.), Gene tube (human MCEMP1 / tube) prepared above fixed to the gun at a pressure of 400 psi, three times every 7 days DNA vaccine into the peritoneal cavity of mice were shaved, after administered transdermally (plasmid DNA inoculum becomes 2 [mu] g / animal with pure helium gas ) the N2a- human MCEMP1 prepared above, was also evaluated the antitumor effect by respectively transplanting N2a-emp as control cells (prophylactic model). As a control, it was administered five plasmid DNA human MCEMP1 gene is not inserted in the prophylaxis model.
[0084]
　Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. The results of this study, in the prevention models N2a- human MCEMP1, and after 28 days, the control group and the human MCEMP1 plasmid administered group, respectively, 1379Mm 3 , 513Mm 3 , and the noticeable tumor regression in humans MCEMP1 plasmid treated group It was observed. Observation of the course of survival in the prevention model, whereas the all cases died 61 days after the administration in the control group, in humans MCEMP1 plasmid treated group, 60% of the mice were alive. These results, N2A in the prevention model of human MCEMP1, human MCEMP1 plasmid treated group significant antitumor effect compared to the control group was observed. On the other hand, in the prevention model N2a-emp, human MCEMP1 plasmid treated group significant antitumor effect compared to the control group was observed.
Industrial Applicability
[0085]
　The present invention is to provide an immunity-inducing agent comprising a polypeptide exhibiting an anti-tumor activity against various cancers, it is useful for the treatment and / or prevention of cancer.
[0086]
　All publications cited herein, patents and patent applications are intended to directly incorporated herein by reference.
The scope of the claims
[Requested item 1]
　The following (a) ~ selected from any of the polypeptides of (d), and at least one polypeptide, or a polynucleotide encoding the polypeptide, and the polypeptide in vivo having an immunity-inducing activity immunity-inducing agent containing a recombinant vector, as an active ingredient which is capable of expressing.
(A) a polypeptide consisting of SEQ ID NO: 2, 4, 6, or fewer amino acid full-length from less than 7 consecutive amino acid sequence shown in 8 of the sequence listing
SEQ ID NO: (b), the sequence listing 2,4,6 , or one to several amino acids in the amino acid sequence shown in 8 by deletion, polypeptides consisting of substituted or added in the amino acid sequence
amino acids shown in SEQ ID NO: 2, 4, 6, or 8 (c) the sequence listing a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to SEQ
polypeptide comprising any of the polypeptides; (d) (a) ~ (c) as a partial sequence
[Requested item 2]
　A treatment agent of antigen-presenting cells, immunity-inducing agent according to claim 1.
[Requested item 3]
　As an active ingredient of the cancer therapeutic and / or prophylactic agent, the immunity-inducing agent according to claim 1.
[Requested item 4]
　Wherein the cancer is a cancer expressing MCEMP1, immunity-inducing agent according to claim 3.
[Requested item 5]
　Wherein the cancer is leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma or perianal adenocarcinoma, immunity-inducing agent according to claim 3 or 4.
[Requested item 6]
　Further comprising an immune enhancer, immunity-inducing agent according to any one of claims 1 to 5.
[Requested item 7]
　The immunopotentiating agent, Freund's incomplete adjuvant, Montanide, poly IC and a derivative thereof, CpG oligonucleotide, interleukin 12, interleukin 18, interferon alpha, interferon beta, interferon omega, from the group consisting of interferon-γ and Flt3 ligand at least one is, the immunity-inducing agent according to claim 6 which is selected.
[Requested item 8]
　An antigen presenting cell-derived polypeptide and a subject as defined in claim 1 comprising contacting ex vivo or in vitro, method for producing antigen-presenting cell comprising a complex between said polypeptide and the MHC molecule.
[Requested item 9]
　Wherein the antigen presenting cells are dendritic cells or B cells bearing MHC class I molecules The method of claim 8.
[Requested item 10]
　Poly and T cells from antigen-presenting cells and a subject obtained by the method according to claim 8 or 9 in contact with ex vivo or in vitro comprising activating the T cells, as defined in claim 1 the method for manufacturing a cytotoxic T cells specific to the peptide.
[Requested item 11]
　Is selected from a polypeptide of the following (a) ~ (d), and at least one polypeptide having immunity-inducing activity, or a polynucleotide encoding said polypeptide and capable of expressing said polypeptide in vivo comprising administering a recombinant vector, to a subject is immune induction method.
(A) SEQ ID NO: 2, 4, 6, or a polypeptide comprising the following amino acid full-length of seven or more consecutive amino acid sequence shown in 8 of the sequence listing
SEQ ID NO: (b), the sequence listing 2,4,6 , or in the amino acid sequence shown in 8, 1 to several amino acids are deleted, a polypeptide consisting of a substituted or added in the amino acid sequence
shown in SEQ ID NO: 2, 4, 6, or 8 (c) the sequence listing a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence
polypeptides comprising; (d) any of the polypeptides of (a) ~ (c) as a partial sequence