RU2777992C2 - Immune-inducing agent - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to an immune-inducing composition containing polypeptide MCEMP1 with an amino acid sequence with SEQ ID NO: 2 or 8; it can be used in medicine.

EFFECT: obtained composition can be used for the effective therapy of MCEMP1-expressing cancer.

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Description

The field to which the invention relates

[0001] The present invention relates to a novel immunoinducing agent that is used as an agent for treating and/or preventing cancer or the like.

Prerequisites for the creation of the invention

[0002] Cancer is the leading cause of death worldwide. Currently, the main method of cancer treatment is surgery, which is carried out in combination with radiation therapy and chemotherapy. In recent years, despite the development of new surgical technologies and the emergence of new anti-cancer agents, cancer treatment, with the exception of certain types of cancer, still does not give the desired results. For example, in recent years, cancer antigens recognized by cytotoxic T cells that react with cancer antigens and genes encoding cancer antigens have been identified, and molecular biology and cancer immunology methods have been developed, which improves the prospects for antigen-specific immunotherapy.

[0003] Mast cell-expressed membrane protein 1 (MCEMP1), i.e. type 2 transmembrane protein, has been reported to be expressed on the cell membrane in a mast cell-specific mechanism, suggesting that this protein is likely to be involved in mast cell differentiation and in inducing an immune response and an allergic response (Non-Patent Document 1). However, there is no evidence in the literature that the MCEMP1 protein has immunoinducing activity against cancer cells and can be used for the treatment and prevention of cancer.

Prior Art

Non-Patent Documents

[0004] Non-Patent Document 1: Kang Li. et al. Genomics 86:68-75(2005)

The essence of the invention

Problems that can be solved with the present invention

[0005] The purpose of the present invention is to discover a new polypeptide that can be used as a therapeutic and/or prophylactic agent for the treatment of cancer, and the use of such a polypeptide as an immunoinducing agent.

Solutions to the problem

[0006] Intensive studies have been carried out by the inventors of the present invention, as a result of which a cDNA encoding a protein that binds to an antibody present in the serum of living individuals with a cancer tumor was obtained by the SEREX method using a cDNA library isolated from the testicles of dogs, as well as in serum dogs with leukemia. Based on this cDNA, the present inventors have obtained a canine mast cell membrane protein 1 (hereinafter referred to as MCEMP1) polypeptide having the amino acid sequence shown in SEQ ID NO: 4. In addition, based on human, feline and murine genes, homologous to the canine gene, human, feline and mouse MCEMP1 polypeptides having the amino acid sequences shown in SEQ ID NOs: 2, 6 and 8 were obtained by the present inventors. The present inventors found that these MCEMP1 polypeptides are specifically expressed in leukemia, myelodysplastic syndrome , osteosarcoma, thymoma, mastocytoma and perianal adenocarcinoma. In addition, we have also found that immune cells directed against MCEMP1 can be induced in vivo by administering these MCEMP1 to living individuals, and that tumor size in living individuals expressing MCEMP1 can be reduced. In addition, the present inventors have found that a recombinant vector capable of expressing a polynucleotide encoding an MCEMP1 polypeptide or a fragment thereof has an antitumor effect on a cancer expressing MCEMP1 in vivo .

[0007] The present inventors have also found that the MCEMP1 polypeptide is presented by an antigen-presenting cell and has the ability (also referred to as "immunoinducing activity") to activate and proliferate cytotoxic T cells specific for this polypeptide; and that due to this ability, this polypeptide can be used for the treatment and/or prevention of cancer, and also that antigen-presenting cells that are in contact with this polypeptide and T-cells that are in contact with an antigen-presenting cell can be used to treat and /or cancer prevention. Based on these data, the present invention was developed.

[0008] Accordingly, the present invention includes the following features (1)-(11):

(1) An immunoinducing agent comprising, as an active ingredient, at least one polypeptide having immunoinducing activity and selected from any of the following polypeptides (a) to (d), or a recombinant vector containing a polynucleotide encoding a polypeptide and capable of expressing said polypeptide in vitro ;

(a) a polypeptide consisting of 7 or more consecutive amino acids or having the full length amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing;

(b) a polypeptide consisting of an amino acid sequence obtained by deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing;

(c) a polypeptide consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing;

(d) a polypeptide containing any of the polypeptides (a)-(c) as a partial sequence.

(2) The immunoinducing agent according to (1), which is an agent for treating antigen presenting cells.

(3) An immunoinducing agent according to (1), which is an active ingredient for the treatment and/or prevention of cancer.

(4) The immunoinducing agent according to (3), wherein the cancer is an MCEMP1-expressing cancer.

(5) The immunoinducing agent according to (3) or (4), wherein the cancer is leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma, or perianal adenocarcinoma.

(6) An immunoinducing agent according to any of (1) to (5), also containing an immunostimulant.

(7) The immunoinducing agent according to (6), wherein the immunostimulant is at least one agent selected from the group consisting of incomplete Freund's adjuvant, montanide, Poly-IC and its derivatives, CpG oligonucleotides, interleukin 12, interleukin 18, interferon α, interferon β, interferon ω, interferon γ, and Flt 3 ligand.

(8) A method for producing an antigen presenting cell comprising a complex of a polypeptide defined in (1) and an MHC molecule, wherein said method comprises contacting the polypeptide with an antigen presenting cell taken from an individual ex vivo or in vitro .

(9) The method according to (8), wherein the antigen presenting cell is a dendritic cell or a B cell containing an MHC class I molecule.

(10) A method for producing a cytotoxic T cell specific for a polypeptide defined in (1), wherein said method comprises contacting an antigen presenting cell obtained by the method according to (8) or (9) with a T cell taken from a subject ex vivo or in vitro , and thereby T-cell activation.

(11) A method for inducing an immune response, comprising administering to an individual at least one polypeptide having immunoinducing activity and selected from any of the following polypeptides (a) to (d), or a recombinant vector containing a polynucleotide encoding a polypeptide and capable of expressing said polypeptide in vivo ;

(a) a polypeptide consisting of 7 or more consecutive amino acids or having the full length amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing;

(b) a polypeptide consisting of an amino acid sequence obtained by deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing;

(c) a polypeptide consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing;

(d) a polypeptide containing any of the polypeptides (a)-(c) as a partial sequence.

Accordingly, the present invention relates to a novel immunoinducing agent that can be used for the treatment and/or prevention of cancer and the like. By administering the polypeptide used in the present invention to an individual, immune cells can be induced in vivo and the cancer that is already present can shrink or regress as specifically described in the examples below. Thus, the polypeptide can be used for the treatment and prevention of cancer.

[0009] The description of the present invention includes the contents of Japanese Patent Application No. 2016-064034, the priority of which is claimed in this application.

Brief description of the drawings

[0010] [Fig. 1] This figure shows the expression patterns of the identified MCEMP1 gene in canine tumor tissues. Number 1 indicates patterns of the canine MCEMP1 gene in individual tumor tissues of dogs.

[Fig. 2] This figure shows expression patterns of the MCEMP1 gene in selected human tissues and cancer cell lines. Number 2 indicates expression patterns of the human MCEMP1 gene in selected healthy human tissues; and number 3 indicates expression patterns of the human MCEMP1 gene in selected human cancer cell lines. In this figure, PBMC means peripheral blood mononuclear cells.

[Fig. 3] This figure shows the expression patterns of the identified MCEMP1 gene in selected mouse cancer cell lines. Number 4 indicates expression patterns of the mouse MCEMP1 gene in selected mouse cancer cell lines.

Method for carrying out the invention

[0011] The present invention is described in more detail below.

1. Polypeptide

Since the polypeptide is contained as an active ingredient in the immunoinducing agent of the invention, the present invention includes the polypeptides defined in the following (a)-(d). As used herein, the term "polypeptide" means a molecule formed by a plurality of amino acids that are linked together by a polypeptide bond, and includes not only a polypeptide molecule consisting of a large number of amino acids, but also a small molecule (i.e., an oligopeptide) consisting of a small number of amino acids, or a full-length protein.

(a) A polypeptide consisting of 7 or more consecutive amino acids or having the full length amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing and having immunoinducing activity;

(b) a polypeptide consisting of an amino acid sequence obtained by deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing; and having immunoinducing activity;

(c) a polypeptide consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the sequence listing; and having immunoinducing activity;

(d) a polypeptide containing any of the polypeptides (a)-(c) as a partial sequence and having immunoinducing activity.

[0012] In the present invention, the phrase "having an amino acid sequence" means that the amino acid residues are located in the sequence in a specific order. Accordingly, for example, the term "polypeptide having the amino acid sequence shown in SEQ ID NO: 8" means a polypeptide having a size of 183 amino acid residues and consisting of the amino acid sequence Met, His, Ala, Ser, Ala.. . Gln, Pro, Ser, and Thr as set forth in SEQ ID NO: 8. A "polypeptide having the amino acid sequence set forth in SEQ ID NO: 8" is sometimes simply referred to herein, for example, as "polypeptide of SEQ ID NO: 8". The same applies to the phrase "having a nucleotide sequence". In this expression, the word "having" can be replaced by the phrase "consisting of".

[0013] As used herein, the term "immunoinducing activity" means the ability to induce immune cells in a living body that secrete cytokines such as interferon.

[0014] The immunoinducing activity of the above polypeptide can be confirmed, for example, using an ELISPOT assay known to those skilled in the art. More specifically, immunoinducing activity can be assessed by obtaining cells, such as peripheral blood mononuclear cells, from a living body into which the polypeptide being evaluated for immunoinducing activity has been introduced; co-culturing these cells with the polypeptide; and measuring the amount of cytokine produced by these cells using a specific antibody, and thereby determining the number of immune cells in these cells.

[0015] As described in the examples below, the introduction of recombinant polypeptides in accordance with (a)-(d) in living organisms with a cancerous tumor can also lead to tumor regression due to the immunoinducing activity of the polypeptides. Accordingly, an immunoinducing activity can be measured by its ability to suppress the proliferation of cancer cells or reduce the size of the cancer tissue (tumor) or even remove the cancer tissue (tumor) (such ability will hereinafter be referred to as "antitumor activity"). The antitumor activity of a polypeptide can actually be confirmed by introducing the polypeptide into a living body with a cancerous tumor, and evaluating, for example, a reduction in tumor size or the absence of such a reduction, for example, as specifically described below in the examples. Alternatively, the antitumor activity of a polypeptide can be assessed to determine, for example, whether a cytotoxic T cell induced by introducing the polypeptide into a living organism with a cancer can have a cytotoxic effect on that tumor. The cytotoxic activity of a T cell can be determined in vivo by administering an antibody that removes the T cell from a living organism and assessing whether or not the tumor has increased in size. However, the method for determining the cytotoxic activity is not limited to the above method.

[0016] Alternatively, the antitumor activity of polypeptides can be assessed by the ability of T cells stimulated with polypeptides (more specifically, T cells in contact with antigen presenting cells that present polypeptides) to exert a cytotoxic effect on tumor cells in vitro . T cells and antigen presenting cells can be brought into contact with each other by co-culturing both cells in a liquid medium as described below. Cytotoxic activity can be assessed by a known method called the ⁵¹ Cr release assay, for example as described in Int. J. Cancer, 58: p. 317, 1994. When the aforementioned polypeptides are used for the treatment and/or prevention of cancer, the immunoinducing activity is preferably measured by antitumor activity serving as an indicator, although such evaluation is not limited to the aforementioned evaluation.

[0017] In the present invention, the amino acid sequences shown in SEQ ID NOs: 2, 4, 6 and 8, respectively, in the sequence listing are MCEMP1 amino acid sequences that have been isolated as polypeptides that bind to specific antibodies present in serum cancerous dogs by the SEREX method using a cDNA library obtained from canine testicles and from the sera of cancerous dogs, as well as homologues isolated from humans, cats and mice (see example 1). Human MCEMP1, which is a human homologue homologous to canine MCEMP1, has a 70% identical nucleotide sequence and 51% amino acid sequence identity. Feline MCEMP1, which is a feline homologue, has 83% identical nucleotide sequence and 64% amino acid sequence identity. Mouse MCEMP1, which is a mouse homologue, has a 65% identical nucleotide sequence and a 47% identical amino acid sequence.

[0018] The polypeptide defined in (a) above is a polypeptide that consists of 7 or more, and preferably 8, 9, or 10 or more consecutive amino acids in a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 and which has immunoinducing activity. It is particularly preferred that the polypeptide has the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8. As is known to those skilled in the art, if a polypeptide has approximately 7 or more amino acid residues, then such a polypeptide may be angiogenic and immunogenic. Thus, if a polypeptide consists of 7 or more consecutive amino acid residues, or contains all amino acid residues (full length sequence) in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8, then it may have immunoinducing activity, and such thus, can be used to obtain an immunoinducing agent according to the invention.

[0019] The principle of inducing an immune response by introducing an antigenic cancer polypeptide is known and is as follows: the polypeptide is introduced into antigen-presenting cells, and then these polypeptides are decomposed into smaller fragments by cellular peptidases, followed by presentation of these fragments on the cell surface. These fragments are then recognized by a cytotoxic T cell or the like, resulting in the selective killing of antigen presenting cells. The polypeptide present on the surface of the antigen-presenting cell is relatively small and consists of approximately 7-30 amino acids. Therefore, in terms of the presentation of the polypeptide on the surface of the antigen presenting cell, one of the preferred variants of the above described polypeptide (a) is a polypeptide consisting of approximately 7-30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 , and more preferably, as the polypeptide (a) can be considered a polypeptide consisting of about 8-30 or about 9-30 amino acids. In some cases, these relatively small polypeptides are presented directly on the surface of the antigen-presenting cell, but do not penetrate into these antigen-presenting cells.

[0020] In addition, since the polypeptide introduced into the antigen-presenting cell is cleaved at non-specific sites by cellular peptidases to form various polypeptide fragments, which are then presented on the surface of the antigen-presenting cell, the introduction of a large polypeptide, such as the full-length region in SEQ ID NO: 2, 4, 6 or 8 inevitably results in the production of polypeptide fragments by cleavage in the antigen presenting cell, and these fragments are effective in inducing an immune response by the antigen presenting cell. Therefore, to induce an immune response by antigen presenting cells, a large polypeptide can preferably be used, which can be at least 30, preferably at least 100, and more preferably at least 200 amino acids. More preferably, such a polypeptide consists of the full length region of SEQ ID NO: 2, 4, 6 or 8.

[0021] The polypeptide described in (c) above is a polypeptide obtained by substitution, deletion and / or insertion of a small number (preferably one or more) amino acid residues in the polypeptide described in (a) above, and such a sequence is 90 % or more, preferably 95% or more, more preferably 98% or more, even more preferably 99% or more or 99.5% or more identical to the original sequence and has immunoinducing activity. Generally speaking, it is well known to those skilled in the art that a protein antigen, even with a substitution, deletion, or insertion of a small number of amino acid residues in the amino acid sequence of a protein, can be substantially as antigenic as the original protein. Accordingly, the polypeptide defined in (c) above may have an immunoinducing activity, and therefore, it can be used to obtain an immunoinducing agent according to the invention. Also preferably, the polypeptide defined in (b) above is a polypeptide obtained by substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8. Used here the term "several" means an integer from 2 to 10, preferably from 2 to 6, and more preferably from 2 to 4.

[0022] As used herein, the term "amino acid sequence or nucleotide sequence identity" means a value calculated by aligning two amino acid sequences (or nucleotide sequences) such that the number of amino acid residues (or nucleotides) that match between amino acid sequences (or nucleotide sequences) is possibility, as high as possible, and then the number of matching amino acid residues (or the number of matching nucleotides) is divided by the total number of amino acid residues (or the total number of nucleotides), and this value is expressed as a percentage. When performing an alignment, if necessary, a space(s) is introduced into one or both of the compared sequences. Such sequence alignment is carried out using a well-known program such as BLAST, FASTA or CLUSTAL W. When a space(s) is entered, the above total number of amino acid residues means the number of residues calculated by counting one space as one amino acid residue. If the thus calculated total numbers of amino acid residues in the two compared sequences differ, then the sequence identity (%) is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues in the longer sequence.

[0023] The 20 types of amino acids that make up natural proteins can be divided into groups according to their similar properties, for example, neutral amino acids with low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids having 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 in most cases, substitutions of amino acids belonging to the same group do not lead to a change in the properties of the polypeptide. Consequently. in the case of substitution of amino acid(s) residue(s) in the polypeptide (a) according to the invention, the probability of retaining immunoinducing activity is increased due to the substitution of amino acid residues belonging to the same group, and such a substitution is preferred.

[0024] The polypeptide (d) includes any of the polypeptides (a)-(c) in a partial sequence, and has immunoinducing activity. That is, the polypeptide (a) or (b) has another amino acid or another polypeptide attached to one end or both ends of any of the polypeptides (a) to (c) and has immunoinducing activity. Such a polypeptide may also be used to produce an immunoinducing agent of the invention.

[0025] The above-described polypeptides can be synthesized, for example, by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (tert-butyloxycarbonyl method). In addition, they can be synthesized by standard methods using various types of commercially available peptide synthesizers. In addition, the polypeptide of interest can be obtained by known genetic engineering techniques by synthesizing a polynucleotide encoding the above polypeptide and incorporating the polynucleotide into an expression vector, which is then introduced into a host cell to produce the polypeptide in that cell.

[0026] A polynucleotide encoding the above polypeptide can be easily obtained by a known genetic engineering method or a standard method using a commercially available nucleic acid synthesizer. For example, DNA having the nucleotide sequence shown in SEQ ID NO: 3 can be obtained by PCR using a canine chromosome DNA or cDNA library as a template and a pair of primers designed so that the nucleotide sequence shown in SEQ ID NO: 3 could be amplified using the primers. DNA having the nucleotide sequence of SEQ ID NO: 1 can be similarly prepared using a human chromosome DNA library or cDNA as a template. The PCR reaction conditions can be selected as appropriate, and examples of such reaction conditions include, but are not limited to: 30 reaction cycles at 94°C, 30 seconds (denaturation), at 55°C, 30 seconds to 1 minute (annealing) , and 1 cycle at 72°C for 1 minute (lengthening), and then the reaction at 72°C for 7 minutes. In addition, the desired DNA can be isolated by obtaining the appropriate probe(s) or primer(s) based on the nucleotide sequence and amino acid sequence information provided in SEQ ID NOs: 1 and 3 in the sequence listing provided herein. , and screening a human, canine, or the like cDNA library. using probe(s) or primer(s). The cDNA library is preferably obtained from cells, organs, or tissues expressing the protein of SEQ ID NO: 2 or 4. The above procedures, such as obtaining the probe(s) or primer(s), constructing the cDNA library, screening the cDNA library, and cloning of interest the genes are known to those skilled in the art and can be carried out using the methods described in Molecular Cloning, Second Edition; Current Protocols in Molecular Biology; and/or the like. From the DNA thus obtained, the DNA encoding the polypeptide (a) can be isolated. In addition, since the codons encoding each amino acid are known, the nucleotide sequence of a polynucleotide encoding a particular amino acid sequence can be easily determined. Therefore, since the nucleotide sequence of a polynucleotide encoding polypeptide (b)-(d) can also be easily determined, such a polynucleotide can also be easily synthesized using a commercially available nucleic acid synthesizer by a standard method.

[0027] Host cells are not particularly limited as long as they can express the above-described polypeptide, and examples of such cells include, but are not limited to, prokaryotic cells such as E. coli ; and eukaryotic cells such as cultured mammalian cells, including COS1 monkey kidney cells and CHO Chinese hamster ovary cells; yeast that reproduces by budding; yeast that reproduces by fission; silkworm cells and Xenopus laevis ovules.

[0028] When using prokaryotic cells as host cells, an expression vector is used in which an origin is present, allowing the vector to replicate in the prokaryotic cell; promoter; ribosome binding site; DNA cloning site; terminator and/or the like. Examples of expression vectors for E. coli are the pUC system, pBluescript II, the pET expression system, and the pGEX expression system. By incorporating DNA encoding the above polypeptide into such an expression vector and transforming prokaryotic host cells with this vector, followed by culturing the resulting transformants, the polypeptide encoded by the DNA can be expressed in prokaryotic host cells. In this method, the polypeptide may also be expressed as a fusion protein attached to another protein.

[0029] When eukaryotic cells are used as host cells, the expression vector is a eukaryotic cell expression vector having a promoter, a splicing region, a poly(A) attachment site, and/or the like. Examples of such an expression vector are pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag (A, B, C), pMSG and pYES2. In a similar method as described above, by incorporating DNA encoding the above polypeptide into such an expression vector, and transforming eukaryotic host cells with this vector, and then culturing the resulting transformants, the polypeptide encoded by the DNA can be expressed in eukaryotic host cells. When pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like are used as an expression vector, the above polypeptide can be expressed as a fusion protein to which labels such as His-tag, FLAG-tag, myc-tag, HA-tag or GFP.

[0030] Introduction of the expression vector into host cells can be carried out by well-known methods such as electroporation, calcium phosphate method, liposomal method and DEAE-dextran method.

[0031] Isolation and purification of a polypeptide of interest from host cells can be accomplished using a combination of known separation techniques. Examples of known separation methods include, but are not limited to, treatment with a denaturing agent such as urea or a surfactant; ultrasonic treatment; enzymatic cleavage; salting out or fractionated solvent precipitation; dialysis; centrifugation; ultrafiltration; gel filtration; electrophoresis in SDS-PAGE; isoelectric focusing; ion exchange chromatography; hydrophobic chromatography; affinity chromatography; and reverse phase chromatography.

[0032] The polypeptides obtained by the method described above also include, as described above, the polypeptides in the form of a hybrid protein along with any other protein. Examples of such proteins are glutathione S-transferase (GST) or His-tag fusion proteins. Such a polypeptide in the form of a fusion protein is also within the scope of the present invention as the above polypeptide (d). In addition, in some cases, the polypeptide expressed in the transformed cell is modified by various methods after it has been translated into the cell. Such a post-translationally modified polypeptide is also within the scope of the present invention, provided that it has immunoinducing activity. Examples of such post-translational modifications are N-terminal methionine removal, N-terminal acetylation, glycosylation, limited intracellular protease cleavage, myristoylation, isoprenylation, and phosphorylation.

[0033] 2. Immunoinducing agent

As described in more detail in the examples below, the introduction of a polypeptide having immunoinducing activity into a living organism with a tumor can cause tumor regression. Thus, the immunoinducing agent according to the invention can be used for the treatment and/or prevention of cancer. In addition, a polypeptide having immunoinducing activity can be used in a method for treating and/or preventing cancer by inducing an immune response.

[0034] As used herein, the terms "tumor" and "cancer" refer to malignant neoplasms and are synonymous.

[0035] In this case, a non-limiting example of a target cancer is any cancer that expresses MCEMP1, and preferably a cancer that expresses MCEMP1 in a significantly higher amount than normal cells, and such cancers are, in particular, leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma, or perianal adenocarcinoma. Specific examples of such cancers include, but are not limited to, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocythemic leukemia, basophilic leukemia, blast leukemia, bovine leukemia, chronic myeloid leukemia, limited leukemia, fetal leukemia, eosinophilic leukemia, Gross leukemia, Reeder's cellular leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cellular leukemia, reticuloendotheliosis, hemoblast leukemia, hemocytoblast leukemia, histiocytic leukemia, stem cell leukemia , acute monocytic leukemia, leukopenia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphotropic leukemia, lymphoid leukemia, lymphosarcoma, cellular leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myelobl myeloid leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Nigeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blast cell (RAEB), RAEB with transformation (RAEB-T), preleukemia and chronic myelomonocytic leukemia (CMML), standard central osteosarcoma and its subtypes (intraosseous well-differentiated osteosarcoma, round cell osteosarcoma, superficial osteosarcoma, periosteal osteosarcoma, periosteal osteosarcoma and highly malignant superficial osteosarcoma), thymoma, mastocytoma, perianal adenoma, and perianal adenocarcinoma.

[0036] The individual (i.e., animal) of interest is preferably a mammal, and more preferably, primates, pets, any animals kept in zoos or the like, farm animals, and animals participating in sports, and especially preferred are human, dogs or cats.

[0037] The method of administering the immunoinducing agent of the invention to a living organism may be oral administration or parenteral administration, and preferably parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, or intra-arterial administration. If an immunoinducing agent is used to treat cancer, it may be injected into a regional lymph node in close proximity to the tumor being treated in order to enhance anti-cancer activity. The dose may be any dose as long as it is effective to induce an immune response, for example, in the case where such an agent is used to treat and/or prevent cancer, then such a dose may be a dose effective to treat and/or prevent cancer. . The dose effective for the treatment and/or prevention of cancer is appropriately selected depending on the size and symptoms of the tumor and the like, and the effective dose is usually 0.0001 μg to 1000 μg, and preferably 0.001 μg to 1000 μg per animal per day, and such dose may be administered as a single dose or divided doses. This agent is preferably administered several times, for example over several days or several months. As described in the examples below, the immunoinducing agent of the invention can cause tumor regression. Therefore, since the agent can have an anti-cancer effect on a small number of cancer cells at an early stage, the development or recurrence of cancer can be prevented using the agent administered before the development of cancer or after its treatment. Thus, the immunoinducing agent of the invention is effective for the treatment and prevention of cancer.

[0038] The immunoinducing agent of the invention may be composed of the polypeptide(s) alone, or it may be formulated by appropriate mixing of additives, such as a pharmacologically acceptable carrier, diluent, excipient, and the like, suitable for the preparation of dosage forms. The method of preparation of the preparation, as well as suitable additives, is well known to those skilled in the art of preparing pharmaceutical preparations, and any methods and any additives can be used. Examples of additives include, but are not limited to, diluents such as physiological buffer solutions; fillers such as sugar, lactose, cornstarch, calcium phosphate, sorbitol and glycine; binding agents such as syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride and tragacanth; and lubricants such as magnesium stearate, polyethylene glycol, talc, and silica. Examples of dosage forms may be oral preparations such as tablets, capsules, granules, powders and syrups; and parenteral preparations such as inhalers, injections, suppositories and solutions. These preparations can be obtained by methods generally known in the art.

[0039] The immunoinducing agent of the invention can be used in combination with an immunostimulant that enhances the immune response in a living organism. The immunostimulant may be contained in the immunoinducing agent of the invention, or it may be administered to the patient as a separate composition in combination with the immunoinducing agent of the invention.

[0040] Examples of immunostimulants are adjuvants. Adjuvants can enhance the immune response by creating an antigen reservoir (outside the cells or in macrophages), activating macrophages, and stimulating specific sets of lymphocytes, which will lead to enhanced immune response and anticancer activity. Therefore, in cases where the immunoinducing agent according to the invention is used for the treatment and/or prevention of cancer, such an immunoinducing agent, in addition to the above-described polypeptide, which is an active ingredient, preferably contains an adjuvant. Many types of adjuvants are well known to those skilled in the art and any of these adjuvants may be used. Specific examples of adjuvants are MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide homologues obtained after purification and acid hydrolysis of lipopolysaccharide; QS21 (SmithKline Beecham), pure QA-21 saponin isolated from Quillja saponaria extract; DQS21 as described in PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So and 10 others., Molecules and Cells, 1997, Vol. 7, p. 178-186); incomplete Freund's adjuvant; complete Freund's adjuvant; vitamin E; Montanid; alum; CpG oligonucleotides (see, for example, Kreig and 7 others, Nature, Vol. 374, p. 546-549); poly-IC and their derivatives (eg poly-ICLC); and various water-in-oil emulsions derived from biodegradable oils such as squalene and/or tocopherol. Of these, Freund's incomplete adjuvant is preferred; Montanid; poly-IC and their derivatives, as well as CpG oligonucleotides. The ratio of the above adjuvant to the polypeptide in the mixture is usually from about 1:10 to 10:1, preferably from about 1:5 to 5:1, and more preferably about 1:1. However, adjuvants are not limited to the examples described above, and adjuvants known to those skilled in the art, but not described above, may also be used when administering the ^{immunoinducing} agent of the invention (see, for example, Goding, "Monoclonal Antibodies: Principles and Practice, 2nd edition", 1986 ). Methods for preparing mixtures or emulsions of a polypeptide and adjuvants are well known to those skilled in the art of vaccination.

[0041] In addition, in addition to the adjuvants described above, factors that stimulate an immune response of interest can be used as an immunostimulant. Thus, for example, various cytokines having the ability to stimulate lymphocytes or antigen presenting cells can be used as an immunostimulant in combination with an immunoinducing agent according to the invention. A number of such cytokines capable of stimulating an immune response are known to those skilled in the art, and examples of such cytokines include, but are not limited to, interleukin-12 (IL-12), GM-CSF, IL-18, interferon-α, interferon-β, interferon- ω, interferon-γ and Flt 3 ligand, which have been reported to enhance the preventive effect of vaccines. Such factors may be used as an immunostimulant and administered to the patient by addition to an immunoinducing agent of the invention, or they may be administered as a separate composition in combination with an immunoinducing agent of the invention.

[0042] 3. Antigen presenting cells or cytotoxic T cells

The present invention also relates to a method for producing antigen-presenting cells containing a complex of the above polypeptide and MHC molecules, which method includes contacting the polypeptide with an antigen-presenting cell taken from an individual ex vivo or in vitro .

[0043] The present invention also relates to an antigen-presenting cell, characterized in that it contains a complex of the aforementioned polypeptide and an MHC molecule, and was obtained by the described method.

[0044] By contacting the above polypeptide with antigen-presenting cells ex vivo or in vitro , antigen-presenting cells can be made to present the polypeptide. That is, the polypeptides (a)-(d) described above can be used as an agent for treating antigen-presenting cells. In the present specification, examples of antigen-presenting cells that are preferably used are dendritic cells or B cells having an MHC class I molecule. Various MHC class I molecules have been identified and are well known. Human MHC molecules are designated HLA. Examples of HLA class I molecules are HLA-A, HLA-B and 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 and HLA-Cw0602.

[0045] Dendritic cells or B cells carrying an MHC class I molecule can be obtained from peripheral blood by a well known method. For example, tumor-specific dendritic cells can be induced by isolating dendritic cells from bone marrow, umbilical cord blood, or peripheral blood of a patient using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 (or IL-4) and followed by adding the tumor-associated peptide to the culture system.

[0046] With the introduction of an effective amount of such dendritic cells, a response necessary for the treatment of cancer can be generated. The cells used may be bone marrow or umbilical cord blood cells taken from a healthy individual, or bone marrow and peripheral blood, etc. taken from the patient himself. When using autologous patient cells, a high degree of safety can be ensured, and it is believed that serious side effects can be avoided in this case. The peripheral blood or bone marrow can be any fresh sample, a refrigerated sample, or a frozen sample. As with peripheral blood, whole blood can be cultured, or individual leukocyte components can be separated and cultured, and as such are more efficient and therefore preferred. In addition, mononuclear cells can be isolated from the leukocyte components. In the case where the cells are derived from bone marrow or umbilical cord blood, whole cells constituting the bone marrow can be cultured, or mononuclear cells can be isolated and cultured from these cells. Peripheral blood and its leukocyte components and bone marrow cells contain mononuclear cells, hematopoietic stem cells, and immature dendritic cells from which dendritic cells are derived, as well as CD4-positive cells, and the like. As for the cytokines to be used, the production method is not particularly limited, and natural or recombinant cytokines or the like are not particularly limited. can be used in any form, provided that they are safe and have physiological activity. Preferably, the drug, the quality of which is suitable for its use in medicine, is used in the required minimum amount. The concentration of the cytokine(s) to be added is not particularly limited, provided such a concentration will allow the induction of dendritic cells, and generally, the total concentration of the cytokine(s) is preferably about 10-1000 ng/mL, and more preferably, approximately 20-500 ng/ml. The culture can be carried out using a well-known medium commonly used for culturing leukocytes. The culture temperature is not particularly limited as long as it is suitable for proliferation of leukocytes, with a temperature of approximately 37° C., which is the human body temperature, being most preferred. The environment during culture is not particularly limited as long as it is suitable for the proliferation of leukocytes, and the ventilation is preferably carried out at 5% CO ₂ . The culturing time is not particularly limited, provided that this period of time is sufficient to induce the required number of cells, and usually, the culturing period is from 3 days to 2 weeks. With regard to equipment used for cell separation and culture, appropriate equipment can be used, and preferably equipment that is safe for medical use and stable and simple procedures. In particular, with regard to cell culture equipment, not only standard vessels such as petri dishes, flasks or bottles, but also universal type vessels, multi-stage vessels, roller bottles, centrifuge type vessels, culturing in bags, hollow fiber columns, or the like.

[0047] The contacting of the above polypeptide with the antigen-presenting cell ex vivo or in vitro itself can be carried out by a method well known in the art, for example, by culturing the antigen-presenting cell in a culture liquid containing the polypeptide. The concentration of the peptides in the medium is not particularly limited and is typically about 1 to 100 µg/ml, and preferably about 5 to 20 µg/ml. The cell density during culture is not particularly limited, and is usually about 10 ³ to 10 ⁷ cells/ml, and preferably about 5×10 ⁴ to 5×10 ⁶ cells/ml. Cultivation is preferably carried out by routine methods at 37°C in an atmosphere of 5% CO ₂ . The length of the peptide that can be presented by the antigen-presenting cell on the surface of the cells is typically about a maximum of 30 amino acid residues. Accordingly, when an antigen-presenting cell is contacted with a polypeptide ex vivo or in vitro , the polypeptide can be made to be about 30 amino acid residues or less in length, however, the length of the peptide is not limited to this number of residues.

[0048] When an antigen-presenting cell is cultured in the presence of the above-described polypeptide, the peptide is integrated into the MHC molecule of the antigen-presenting cell and presented on the surface of the antigen-presenting cell. Accordingly, an isolated antigen-presenting cell containing a complex of a polypeptide and an MHC molecule can be obtained. Such an antigen-presenting cell can present a polypeptide to a T cell in vivo , ex vivo or in vitro , which will result in the induction and proliferation of cytotoxic T cells specific for the polypeptide.

[0049] The present invention also relates to a method for obtaining a cytotoxic T cell specific for the above polypeptide, which method includes contacting antigen presenting T cells with a T cell taken from an individual, ex vivo or in vitro to activate the T cell.

[0050] The present invention also relates to a cytotoxic T cell specific for the above polypeptide and obtained by this method.

[0051] By contacting an antigen-presenting cell containing the complex of the above polypeptide and the MHC molecule obtained by the above method with a T cell ex vivo or in vitro , a polypeptide-specific cytotoxic T cell can be induced and the cell can be subjected to proliferation. Contacting can be carried out by co-culturing the antigen presenting cell and T cell in a liquid medium, for example, by suspending the antigen presenting cell in a liquid medium, placing the resulting suspension in a container such as microplate wells, adding T cells to the wells and culturing them. The ratio of the mixture of antigen-presenting cells and T cells in co-culture is not particularly limited, and is usually about 1:1 to 1:100, and preferably about 1:5 to 1:20. The density of antigen-presenting cells in the liquid medium is not particularly limited and is usually about 100 to 10,000,000 cells/ml, and preferably about 10,000 to 1,000,000 cells/ml. Co-culture is preferably carried out by routine methods at 37°C in an atmosphere of 5% CO ₂ . The culture time is not particularly limited, and is usually 2 days to 3 weeks, and preferably about 4 days to 2 weeks. Co-culture is preferably carried out in the presence of one or more types of interleukins such as IL-2, IL-6, IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5 to 20 U/ml, the concentration of IL-6 is usually about 500 to 2000 U/ml, and the concentration of IL-12 is usually about 5 up to 20 ng / ml, however, these concentrations are not limited to the indicated concentrations. Co-culturing can be done one or more times by adding fresh antigen-presenting cells. Thus, for example, the procedure of removing the culture supernatant after co-cultivation, adding a suspension of fresh antigen-presenting cells, and conducting co-cultivation may be repeated one or more times. The co-cultivation conditions may be the same as those described above.

[0052] During co-culture, induction and proliferation of a polypeptide-specific cytotoxic T cell is carried out. Accordingly, the above polypeptide can be used to obtain an isolated T cell that selectively binds to a complex of a polypeptide and an MHC molecule.

[0053] As described in the examples below, the MCEMP1 gene is specifically expressed in leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma, or perianal adenocarcinoma. Consistent with this, it is believed that in these cancers, MCEMP1 is present in significantly higher amounts than in normal cells. Accordingly, if a cytotoxic T cell obtained by the above method is administered in vivo and a portion of the MCEMP1 polypeptide present in cancer cells is presented by an MHC molecule on the surface of the cancer cell, the cytotoxic T cell can destroy the cancer cell by using the portion of the polypeptide MCEMP1 as a marker. An antigen presenting cell presenting a portion of an MCEMP1 polypeptide can promote the induction and proliferation of a cytotoxic T cell specific for the polypeptide in vivo . Thus, cancer cells can also be destroyed by introducing an antigen-presenting cell into a living body. More specifically, a cytotoxic T cell and an antigen presenting cell obtained using the above described polypeptide can also be used for the treatment and/or prevention of cancer, in analogy with the immunoinducing agent of the invention.

[0054] In the case where the isolated antigen-presenting cells or isolated T cells described above are introduced into a living body, these cells are preferably obtained by treating antigen-presenting cells or T cells taken from a patient to be treated with polypeptide (a)-( d) as described above to prevent an immune response in vivo attacking these cells as foreign cells.

[0055] An agent for the treatment and/or prevention of cancer, comprising antigen-presenting cells or T cells as an active ingredient, is preferably administered parenterally, for example, intravenously or intra-arterially. The dose is appropriately selected depending on the symptoms, purpose of administration, and the like, but usually such a dose is from 1 cell to 10,000,000,000,000 cells, and preferably from 1,000,000 cells to 1,000,000,000 cells, and such a dose is preferably administered once every few days or once every few months. Such preparation may be, for example, cells suspended in buffered saline, where said preparation may be used in combination with other(s) anticancer(s) agent(s), cytokine(s), or the like. In addition, one or more additives well known to those skilled in the art may also be added.

[0056] 4. DNA vaccine

Expression of a polynucleotide encoding any of the polypeptides (a) to (d) may also induce the production of antibodies and cytotoxic T cells in the animal body, and this effect is comparable to that observed when the polypeptide is administered. That is, the immunoinducing agent of the invention may be an agent containing as an active ingredient a recombinant vector having a polynucleotide encoding any of the polypeptides (a) to (d), wherein such recombinant vector is capable of expressing the polypeptide in a living body. As described in the examples below, such a recombinant vector capable of expressing an antigenic polypeptide is also referred to as a DNA vaccine.

[0057] The vector used to produce the DNA vaccine is not particularly limited, provided that it is a vector capable of expressing the polypeptide in an animal cell (preferably a mammalian cell), and the vector may be a plasmid vector or a viral vector, and any vector known to those skilled in the art of preparing DNA vaccines can be used. A polynucleotide, such as DNA or RNA, encoding the above-described polypeptide can be easily obtained by a standard method as described above. The introduction of the polynucleotide into the vector can be carried out in a manner well known to those skilled in the art.

[0058] The route of administration of the DNA vaccine is preferably parenteral administration such as intramuscular, subcutaneous, intravenous or intra-arterial administration, and the dose may be appropriately selected depending on the type of antigen and the like, but is usually about 0.1 μg to 100 mg, and preferably from about 1 μg to 10 mg by weight of DNA vaccine per 1 kg of body weight.

[0059] Examples of a viral vector method are methods in which a polynucleotide encoding the above-described polypeptide is introduced into an RNA virus or a DNA virus such as a retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, or virus Sindbis, and then the animal is infected with the resulting virus. Of these methods, methods using retrovirus, adenovirus, adeno-associated virus, vaccinia virus or the like are particularly preferable.

[0060] Examples of other methods are methods in which the expression plasmid is injected directly into the muscle (DNA vaccine method), the liposome method, the lipofectin method, the microinjection method, the calcium phosphate method, and the electroporation method, and are especially preferred. DNA vaccine based method and liposomal method.

[0061] Methods for using a gene encoding the above-described polypeptide of the invention as a drug include an in vivo method in which the gene is directly introduced into the body, and an ex vivo method in which certain types of cells are taken from an animal and then the gene is introduced into them. , after which these cells are returned to the individual (Nikkei Science, 1994, April, p. 20-45; The Pharmaceutical Monthly, 1994, Vol. 36, No. 1, p. 23-48; Experimental Medicine, Extra Edition, 1994, Vol. 12, No. 15, and works cited in these publications, etc.). More preferred is the in vivo method.

[0062] When a gene is introduced by an in vivo method, such method of introduction depends on the disease being treated, its symptoms, and the like. The gene may be administered, for example, intravenously, intra-arterially, subcutaneously or intramuscularly. When a gene is introduced by the in vivo method, the gene can be obtained in the form of a preparation such as a solution, but mainly an injection or the like, containing the DNA encoding the above-described polypeptide according to the invention as an active ingredient, and when If necessary, a standard carrier may also be added to this solution. In the case of using a liposome or membrane-bound liposome (e.g., Sendai virus (HVJ)-bound liposome) containing DNA, the liposome can be prepared as a liposome preparation such as a suspension, a frozen preparation, or a frozen centrifuge-concentrated preparation. .

[0063] As used herein, the term "nucleotide sequence shown in SEQ ID NO: 1" includes, for example, not only the nucleotide sequence shown in SEQ ID NO: 1, but also a sequence complementary to that sequence. For example, the term "polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1" includes a single-stranded polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1; single-stranded polynucleotide having a nucleotide sequence complementary to this sequence; and a double-stranded polynucleotide consisting of these single-stranded polynucleotides. To obtain a polynucleotide encoding a polypeptide used in the present invention, any of these nucleotide sequences is appropriately selected, and such a selection procedure can be easily carried out by a person skilled in the art.

Examples

[0064] The present invention is described in more detail below with reference to the Examples. However, the scope of the present invention is not limited to these examples.

[Example 1]

Obtaining a new cancer antigenic protein by the SEREX method

(1) Preparation of cDNA library

Total RNA was extracted from dog testicles using the acid-guanidinium-phenol-chloroform method, and then poly(A) RNA was purified using the Oligotex-dT30 mRNA purification kit (Takara Shuzo Co., Ltd.) according to the protocol attached to set.

[0065] Using the obtained mRNA (5 µg), a phage cDNA library was synthesized. To obtain a phage cDNA library, a cDNA synthesis kit, a Zap cDNA synthesis kit, or a GigapackIII Gold Cloning Kit (STRATAGENE) for ZAP cDNA cloning was used according to the protocol supplied with the kit. The size of the resulting phage cDNA library was 1×10 ⁶ b.o.u./ml.

[0066] (2) Screening cDNA library using serum

Immunological screening was carried out using the obtained phage cDNA library. More specifically, E. coli (XL1-Blue MRF') host cells were infected with phage to produce approximately 2500 clones on a φ90×15 mm NZY agarose plate and cultured at 42° C. for 3-4 hours until plaque formation. The plate was sensitized with a nitrocellulose membrane (Hybond C Extra: GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours to induce protein expression, and the protein was transferred to the membrane. Then, the membrane was wetted in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.5% skim milk powder and shaken at 4° C. overnight to suppress non-specific reaction. This filter was left to react with 500-fold diluted dog serum for 2-3 hours at room temperature.

[0067] In the case of using the above described dog serum, this serum was taken from dogs with leukemia. Serum was stored at -80°C and pre-treated just prior to use. The whey pretreatment method is as follows. First, Escherichia coli (XL1-Blue MRF') host cells were infected with λ ZAP Express phage that had not been introduced with the foreign gene, and then cultured in NZY plate medium at 37° C. overnight. Thereafter, 0.2 M NaHCO ₃ buffer (pH 8.3) containing 0.5 M NaCl was added to the plate, and then the plate was left for 15 hours at 4° C. and the supernatant was collected as Escherichia coli /phage extract. The collected Escherichia coli /phage extract was then passed through an NHS column (GE Healthcare Bio-Science) to immobilize proteins derived from Escherichia coli /phage on the column. Dog serum was passed through the column and reacted with the protein immobilized on the column to remove antibodies adsorbed to Escherichia coli and phage from the column. The serum fraction passed through the column was diluted 500-fold with TBS containing 0.5% skimmed milk powder, and the resulting diluent was used as material for immunological screening.

[0068] The above membrane, which was stained with treated serum and proteins, was washed 4 times with TBS-T (0.05% Tween 20/TBS) and reacted with goat anti-canine IgG (HRP-conjugated goat anti-canine IgG). -h+L; BETHYL Laboratories), 5000-fold diluted TBS containing 0.5% skimmed milk powder, as a "second" antibody at room temperature for 1 hour, and then detected by an enzymatic color reaction using a reaction solution NBT/BCIP (Roche). Colonies at sites where a positive color reaction was observed were taken from an NZY agarose plate having a size of φ90×15 mm and dissolved in 500 μl of SM buffer (100 mM NaCl, 10 mM MgClSO ₄ , 50 mM Tris-HCl, 0 01% gelatin, pH 7.5). This screening was repeated two to three times in the manner described above to generate one positively stained colony, and then, after screening approximately 10,000 IgG-reactive phage clones in serum, one positive clone was isolated.

[0069] (3) Search for identical sequences of an isolated antigenic gene

To analyze the nucleotide sequence of one positive clone isolated by the method described above, a procedure was carried out for converting the phage vector into a plasmid vector. In particular, a solution (200 μl) containing Escherichia coli (XL1-Blue MRF') host cells prepared to have an optical density OD ₆₀₀ =1.0; purified phage solution (100 µl) and another 1 µl ExAssist helper phage (STRATAGENE) were mixed and left for 15 minutes to react at 37°C. Then LB medium (3 ml) was added and cultivation was carried out at 37°C for 2.5-3 hours. The resulting culture was immediately placed in a water bath for 20 minutes at 70°C, and then centrifuged at 4°C at 1000×g for 15 minutes to collect the supernatant as a phagemid solution. Then, a solution (200 μl) containing Escherichia coli (SOLR) phagemid host cells prepared to have an optical density OD ₆₀₀ = 1.0 and a purified phage solution (10 μl) were mixed and left for 15 minutes to react at 37°C. The resulting solution (50 µl) was plated on LB agar medium containing ampicillin (final concentration: 50 µg/ml) and cultured overnight at 37°C. One transformed SOLR colony was harvested, cultured in LB medium containing ampicillin (final concentration: 50 μg/mL) at 37° C., and then purified using the QIAGEN Miniprep Kit (QIAGEN) to obtain plasmid DNA, with the correct insert.

[0070] The purified plasmid was primer-walked using the T3 primer shown in SEQ ID NO: 9 and the T7 primer shown in SEQ ID NO: 10 to analyze the full-length sequence of the insert. The gene sequence shown in SEQ ID NO: 3 was obtained by sequencing analysis. Using the nucleotide sequence of the gene and the amino acid sequence, sequence identity analysis of known genes was performed using the BLAST sequence identity search program (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, the resulting gene was found to be the MCEMP1 gene. In human MCEMP1, which is the human homologue of canine MCEMP1, nucleotide sequence identity is 70% and amino acid sequence identity is 51%. In the feline homologue, ie feline MCEMP1, the nucleotide sequence identity is 83% and the amino acid sequence identity is 64%. In the mouse homologue, ie mouse MCEMP1, the nucleotide sequence identity is 65% and the amino acid sequence identity is 47%. The nucleotide sequence of human MCEMP1 is shown in SEQ ID NO: 1 and its amino acid sequences are shown in SEQ ID NO: 2. The nucleotide sequence of feline MCEMP1 is shown in SEQ ID NO: 5 and its amino acid sequences are shown in SEQ ID NO: 6. Nucleotide sequence murine MCEMP1 is shown in SEQ ID NO: 7 and its amino acid sequences are shown in SEQ ID NO: 8.

[0071] (4) Analysis of gene expression in various tissues

The expression of the genes obtained by the above method in normal tissues, in tumor tissues and in cancer cell lines of dogs, humans and mice was assessed by RT-PCR (reverse transcriptase PCR). The reverse transcription reaction was carried out as described below. First, total RNA was extracted from individual tissues (50-100 mg) and individual cell lines (5-10×10 ⁶ cells) using TRIZOL reagent (Life technology) according to the protocol provided. Using total RNA, cDNAs were synthesized using the Superscript First Strand Synthesis System for RT-PCR (Life technology) according to the protocol provided. As cDNA of normal human tissues (brain, testes, colon and placenta), gene pool cDNA (Life technology), clone QUICK cDNA (Clontech), and large insert cDNA library (Clontech) were used. The PCR reaction was carried out using the obtained gene-specific primers (canine primers are shown in SEQ ID NOS: 11 and 12, human primers are shown in SEQ ID NOS: 13 and 14, and mouse primers are shown in SEQ ID NOS: 15 and 16) as follows. That is, reagents were added to the bound buffer, which contained 0.25 μl of the sample obtained by the reverse transcription reaction, the above-described primers (2 μm each), dNTP (0.2 mM each) and 0.65 units. ExTaq polymerase (Takara Shuzo Co., Ltd.). A total of 25 μl of the reaction mixture was subjected to thermowell PCR (BIO RAD). This PCR was performed 30 cycles, where one cycle consisted of treatment at 94°C for 30 seconds; at 55°C for 30 seconds; and at 72°C for one minute. As a result, as shown in FIG. 1, the canine MCEMP1 gene was highly expressed in canine tumor tissues, ie mastocytoma and perianal adenocarcinoma (FIG. 1). The human MCEMP1 gene was not expressed in almost any of the normal human tissues, but was expressed at a high level in human cancer cells, ie, leukemia, myelodysplastic syndrome, and osteosarcoma cell lines (FIG. 2). In addition, mouse MCEMP1 gene expression was detected in leukemia and thymoma cell lines (FIG. 3).

[0072][Example 2]

MCEMP1 in vivo cancer antigenicity assay

(1) Production of a recombinant vector expressing mouse MCEMP1 in vivo

A recombinant vector expressing mouse MCEMP1 in vivo was generated based on the nucleotide sequence shown in SEQ ID NO: 7 using the method described below. PCR was carried out as follows. The reaction mixture was prepared by adding reagents: cDNA (1 μl) which was obtained from the mouse leukemia cell line EL4 (purchased from ATCC), the expression of which was observed as described in Example 1; primers of two types (0.4 μm each) having sequences cleaved with restriction enzymes EcoRI and NotI (represented by SEQ ID NOS: 17 and 18); 0.2 mM dNTP and 1.25 units. PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.) and bound buffer to give a total of 50 μl of the mixture, which was subjected to thermowell PCR (BIO RAD). This PCR was performed 30 cycles, where one cycle consisted of processing at 98°C for 10 seconds; at 55°C for 15 seconds; and at 72°C for 1 minute. The above two types of primers were used to amplify the region encoding the full length amino acid sequence shown in SEQ ID NO: 8. After PCR, the amplified DNA was subjected to 1% agarose gel electrophoresis, and a DNA fragment of approximately 550 bp. purified using a QIAquick gel extraction kit (QIAGEN).

[0073] The purified DNA fragment was ligated to the pCR-Blunt cloning vector (Life technology), and then this vector was transformed into E. coli cells and the plasmid vector was assembled. Sequencing confirmed that the sequence of the amplified gene fragment was identical to the desired sequence. Plasmid, the sequence of which was identical to the desired sequence, was treated with restriction enzymes EcoRI and NotI. After purification using the QIAquick gel extraction kit, the desired gene sequence was inserted into the pcDNA3.1 mammalian expression vector (Invitrogen) treated with restriction enzymes EcoRI and NotI (hereinafter referred to as mouse MCEMP1/pcDNA3.1). Using a vector, the mouse MCEMP1 protein was produced in a mammalian cell.

[0074] To plasmid DNA (100 μg) obtained as described above, was added 50 μg of gold particles (Bio Rad), spermidine (100 μl) (SIGMA) and 1M CaCl ₂ (100 μl (SIGMA)). The mixture was stirred on a vortex mixer and left for 10 minutes at room temperature (hereinafter, this mixture will be referred to as "golden DNA particles"). After centrifugation at 3000 rpm for one minute, the supernatant was discarded and then the pellet was washed three times with 100% ethanol (WAKO). 100% ethanol (6 ml) was added to the golden DNA particles and the mixture was stirred for a sufficient period of time on a vortex mixer. Gold DNA particles were poured into a system of Tefzel Tubing (Bio Rad) tubes to deposit these particles on the walls of the tubes. The Tefzel Tubing tube system with gold DNA particles deposited thereon was air-dried by removing ethanol, and then the particles were cut into pieces having a length suitable for their use in a gene-shooting device (hereinafter, they will be referred to as murine MCEMP1/tube).

[0075] (2) Antitumor effect of murine MCEMP1 achieved by DNA vaccine-1 method

Five A/J mice (7 week old male purchased from Japan SLC, Inc.) were used. Tubes (mouse MCEMP1/tube) prepared as described above were mounted on the gene-shooting device. The DNA vaccine was administered percutaneously three times over a total of 7 days into the shaved peritoneal cavity of mice by supplying pure helium gas at 400 psi (the amount of plasmid DNA injected was 2 μg/animal). After transdermal administration, cells of the mouse leukemia cell line EL4, which were found to express the MCEMP1 gene as described in Example 1, were transplanted into each mouse to evaluate the antitumor effect (this model is called the prophylactic model). As a control, five mice of the prophylaxis model were injected with plasmid DNA without the inserted mouse MCEMP1 gene.

[0076] The antitumor effect was assessed by tumor size (longer diameter x short diameter ^2/2 ) and survival of mice. As a result of this study, in the prophylactic model, tumor sizes after 28 days in the control group and in the group injected with the mouse plasmid MCEMP1 were 1153 mm ³ and 480 mm ³ , respectively. Thus, it was found that the tumor size of the group injected with the mouse plasmid MCEMP1 was significantly reduced. As a result, in accordance with the survival observed for the prophylactic model, it was found that all animals in the control group died 59 days after administration, and in the group injected with the mouse plasmid MCEMP1, 60% of the mice were alive. Based on these results, it can be said that the antitumor effect of the group injected with the mouse plasmid MCEMP1 was more pronounced than that of the control group.

[0077] (3) Antitumor effect of mouse MCEMP1 achieved by DNA vaccine-2 method

Five A/J mice (7 week old male purchased from Japan SLC, Inc.) were used. Tubes (mouse MCEMP1/tube) prepared as described above were mounted on the gene-shooting device. The DNA vaccine was administered percutaneously three times over a total of 7 days into the shaved peritoneal cavity of mice by supplying pure helium gas at 400 psi (the amount of plasmid DNA injected was 2 μg/animal). After transdermal administration, cells of the mouse leukemia cell line EG7, which were found to express the MCEMP1 gene as described in Example 1, were transplanted into each mouse to evaluate the antitumor effect (this model is called the prophylactic model). As a control, five mice of the prophylaxis model were injected with plasmid DNA without the inserted mouse MCEMP1 gene.

[0078] Antitumor activity was assessed by tumor size (longer diameter × short diameter ^2/2 ) and survival of mice. As a result of this study, in the prophylactic model, tumor sizes after 28 days in the control group and the group injected with the mouse plasmid MCEMP1 were 982 mm ³ and 521 mm ³ , respectively. Thus, it was found that the tumor size of the group injected with the mouse plasmid MCEMP1 was significantly reduced. As a result, in accordance with the survival observed for the prophylactic model, it was found that all animals in the control group died 68 days after administration, and in the group injected with the mouse plasmid MCEMP1, 60% of the mice were alive. Based on these results, it can be said that the antitumor effect of the group injected with the mouse plasmid MCEMP1 was more pronounced than that of the control group.

[0079] (4) Production of a recombinant vector expressing human MCEMP1 in vivo

A recombinant vector expressing human MCEMP1 in vivo was generated based on the nucleotide sequence shown in SEQ ID NO: 1 using the method described below. PCR was carried out as follows. The reaction mixture was prepared by adding reagents: cDNA (1 μl) which was obtained from the human leukemia cell line U937 (purchased from ATCC), the expression of which was observed as described in Example 1; primers of two types (0.4 μm each) having sequences cleaved with restriction enzymes EcoRI and NotI (represented in SEQ ID NO: 19 and 20); 0.2 mM dNTP and 1.25 units. PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.) and bound buffer to give a total of 50 μl of the mixture, which was subjected to thermowell PCR (BIO RAD). This PCR was performed 30 cycles, where one cycle consisted of treatment at 98°C for 10 seconds; at 55°C for 15 seconds; and at 72°C for 1 minute. The above two types of primers were used to amplify the region encoding the full length amino acid sequence shown in SEQ ID NO: 2. After PCR, the amplified DNA was subjected to 1% agarose gel electrophoresis, and a DNA fragment of approximately 550 bp. purified using a QIAquick gel extraction kit (QIAGEN).

[0080] The purified DNA fragment was ligated to the pCR-Blunt cloning vector (Life technology), and then this vector was transformed into E. coli cells and the plasmid vector was assembled. Sequencing confirmed that the sequence of the amplified gene fragment was identical to the desired sequence. Plasmid, the sequence of which was identical to the desired sequence, was treated with restriction enzymes EcoRI and NotI. After purification using the QIAquick gel extraction kit, the desired gene sequence was inserted into the pcDNA3.1 mammalian expression vector (Invitrogen) treated with restriction enzymes EcoRI and NotI (hereinafter referred to as human MCEMP1/pcDNA3.1). Using a vector, the human MCEMP1 protein was produced in a mammalian cell.

[0081] To plasmid DNA (100 μg) obtained as described above, was added 50 μg of gold particles (Bio Rad), spermidine (100 μl) (SIGMA) and 1M CaCl ₂ (100 μl (SIGMA)). The mixture was stirred on a vortex mixer and left for 10 minutes at room temperature (hereinafter, this mixture will be referred to as "golden DNA particles"). After centrifugation at 3000 rpm for one minute, the supernatant was discarded and then the pellet was washed three times with 100% ethanol (WAKO). 100% ethanol (6 ml) was added to the golden DNA particles and the mixture was stirred for a sufficient period of time on a vortex mixer. Gold DNA particles were poured into a system of Tefzel Tubing (Bio Rad) tubes to deposit these particles on the walls of the tubes. The Tefzel Tubing tube system, with gold DNA particles deposited thereon, was air dried by removing ethanol, and then the particles were cut into pieces having a length suitable for their use in a gene-shooting device (hereinafter, they will be referred to as human MCEMP1/tube).

[0082] (5) Generation of cells stably expressing full length human MCEMP1

Human MCEMP1/pcDNA3.1, obtained as described above, was introduced by lipofection into the mouse neuroblastoma cell line N2a (ATCC), and then selected using 500 μg/ml G418 (Nacalai Tesque, Inc.) to obtain an N2a cell line, stably expressing full-length human MCEMP1 (N2a-human MCEMP1). Cells obtained after introducing an expression vector (hereinafter referred to as emp/pcDNA3.1) without an inserted cDNA encoding human MCEMP1, followed by selection as described above, were used as control cells (hereinafter referred to as N2a-emp).

[0083] (6) Antitumor effect of human MCEMP1 achieved by DNA vaccine method

Five A/J mice (7 week old male purchased from Japan SLC, Inc.) were used. Tubes (human MCEMP1/tube) prepared as described above were mounted on the gene-shooting device. The DNA vaccine was administered percutaneously three times over a total of 7 days into the shaved peritoneal cavity of mice by supplying pure helium gas at 400 psi (the amount of plasmid DNA injected was 2 μg/animal). After transdermal administration, N2a-human MCEMP1 or N2a-emp, used as control cells, prepared as described above, was transplanted into each mouse to evaluate the antitumor effect (this model is called the prophylactic model). As a control, 5 mice of the prophylaxis model were injected with plasmid DNA without the inserted human MCEMP1 gene.

[0084] Antitumor activity was assessed by tumor size (longer diameter × short diameter ^2/2 ) and survival of mice. As a result of this study, in the N2a-human MCEMP1 prophylactic model, tumor sizes after 28 days in the control group and the group injected with the human MCEMP1 plasmid were 1379 mm ³ and 513 mm ³ , respectively. Thus, it was found that the tumor size of the group injected with the human MCEMP1 plasmid was significantly reduced. As a result, in accordance with the survival observed for the prophylactic model, it was found that all animals in the control group died 61 days after injection, and in the group that was injected with the human plasmid MCEMP1, 60% of mice were alive. Based on these results, it can be said that the antitumor effect of the N2a-human MCEMP1 prophylactic model group injected with the human MCEMP1 plasmid was more pronounced than that of the control group. On the other hand, no antitumor effect was observed in the N2a-emp prophylactic model group injected with the human MCEMP1 plasmid, as in the control group.

Industrial Application

[0085] The present invention relates to an immunoinducing agent containing a polypeptide having anticancer activity, and therefore, this agent can be used for the treatment and/or prevention of cancer.

[0086] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

--->

SEQUENCE LIST

<110> TORAY INDUSTRIES, INC.

<120> Immunoinducing agent

<130> PH-6863-PCT

<150> JP 2016-064034

<151> 2016-03-28

<160> 20

<170>PatentIn version 3.5

<210> 1

<211> 1326

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (26)..(589)

<400> 1

tggacaaatt tgcgggctgg ggacc atg gaa gtg gag gaa atc tac aag cac 52

Met Glu Val Glu Glu Ile Tyr Lys His

fifteen

cag gaa gtc aag atg caa gca cca gcc ttc agg gac aag aaa cag ggg 100

Gln Glu Val Lys Met Gln Ala Pro Ala Phe Arg Asp Lys Lys Gln Gly

10 15 20 25

gtc tca gcc aag aat caa ggt gcc cat gac cca gac tat gag aat atc 148

Val Ser Ala Lys Asn Gln Gly Ala His Asp Pro Asp Tyr Glu Asn Ile

30 35 40

acc ttg gcc ttc aaa aat cag gac cat gca aag ggt ggt cat tca cga 196

Thr Leu Ala Phe Lys Asn Gln Asp His Ala Lys Gly Gly His Ser Arg

45 50 55

ccc acg agc caa gtc cca gcc cag tgc agg ccg ccc tca gac tcc acc 244

Pro Thr Ser Gln Val Pro Ala Gln Cys Arg Pro Ser Asp Ser Thr

60 65 70

cag gtc ccc tgc tgg ttg tac aga gcc atc ctg agc ctg tac atc ctc 292

Gln Val Pro Cys Trp Leu Tyr Arg Ala Ile Leu Ser Leu Tyr Ile Leu

75 80 85

ctg gcc ctg gcc ttt gtc ctc tgc atc atc ctg tca gcc ttc atc atg 340

Leu Ala Leu Ala Phe Val Leu Cys Ile Ile Leu Ser Ala Phe Ile Met

90 95 100 105

gtg aag aat gct gag atg tcc aag gag ctg ctg ggc ttt aaa agg gag 388

Val Lys Asn Ala Glu Met Ser Lys Glu Leu Leu Gly Phe Lys Arg Glu

110 115 120

ctt tgg aat gtc tca aac tcc gta caa gca tgc gaa gag aga cag aag 436

Leu Trp Asn Val Ser Asn Ser Val Gln Ala Cys Glu Glu Arg Gln Lys

125 130 135

aga ggc tgg gat tcc gtt cag cag agc atc acc atg gtc agg agc aag 484

Arg Gly Trp Asp Ser Val Gln Gln Ser Ile Thr Met Val Arg Ser Lys

140 145 150

att gat aga tta gag acg aca tta gca ggc ata aaa aac att gac aca 532

Ile Asp Arg Leu Glu Thr Thr Leu Ala Gly Ile Lys Asn Ile Asp Thr

155 160 165

aag gta cag aaa atc ttg gag gtg ctg cag aaa atg cca cag tcc tca 580

Lys Val Gln Lys Ile Leu Glu Val Leu Gln Lys Met Pro Gln Ser Ser

170 175 180 185

cct caa taa atgagaggac attgtggcag ccaaagccac aacttggaag 629

ProGln

atggggctgc acctgccaac gaagacggga aatgaccccc cccccccagc ctagtgtgaa 689

cctgcccctc gtcccacgta tagaaaaacc tcgagtcatg gtgaatgagt gtctcggagt 749

tgctcgtgtg tgtgtacacc tgcgtgcgtg tgtgtgcgtg tgtgcgcgtg tgttcgtgta 809

tgtgcgtgtg tgcgtgcgcg tgtgtgtgca ttttgcaaag ggtggacatt tcagtgtatc 869

tcccagaaag gtgatgaatg aataggactg agagtcacag tgaatgtggc atgcatgcct 929

gtgtcatgtg acatatgtga gtctcggcat gtcacggtgg gtggctgtgt ctgagcacct 989

ccagcagatg tcactctgag tgtgggtgtt ggtgacatgc attgcacgggg cctgtctccc 1049

tgtttgtgta aacatactag agtatactgc ggcgtgtttt ctgtctaccc atgtcatggt 1109

gggggagatt tatctccgta catgtgggtg tcgccatgtg tgccctgtca ctatctgtgg 1169

ctgggtgaac ggctgtgtca ttatgagtgt gccgagttat gccaccctgt gtgctcaggg 1229

cacatgcaca cagacattta tctctgcact cacattttgt gacttatgaa gataaataaa 1289

1326

<210> 2

<211> 187

<212> PRT

<213> Homo sapiens

<400> 2

Met Glu Val Glu Glu Ile Tyr Lys His Gln Glu Val Lys Met Gln Ala

1 5 10 15

Pro Ala Phe Arg Asp Lys Lys Gln Gly Val Ser Ala Lys Asn Gln Gly

20 25 30

Ala His Asp Pro Asp Tyr Glu Asn Ile Thr Leu Ala Phe Lys Asn Gln

35 40 45

Asp His Ala Lys Gly Gly His Ser Arg Pro Thr Ser Gln Val Pro Ala

50 55 60

Gln Cys Arg Pro Ser Asp Ser Thr Gln Val Pro Cys Trp Leu Tyr

65 70 75 80

Arg Ala Ile Leu Ser Leu Tyr Ile Leu Leu Ala Leu Ala Phe Val Leu

85 90 95

Cys Ile Ile Leu Ser Ala Phe Ile Met Val Lys Asn Ala Glu Met Ser

100 105 110

Lys Glu Leu Leu Gly Phe Lys Arg Glu Leu Trp Asn Val Ser Asn Ser

115 120 125

Val Gln Ala Cys Glu Glu Arg Gln Lys Arg Gly Trp Asp Ser Val Gln

130 135 140

Gln Ser Ile Thr Met Val Arg Ser Lys Ile Asp Arg Leu Glu Thr Thr

145 150 155 160

Leu Ala Gly Ile Lys Asn Ile Asp Thr Lys Val Gln Lys Ile Leu Glu

165 170 175

Val Leu Gln Lys Met Pro Gln Ser Ser Pro Gln

180 185

<210> 3

<211> 1124

<212> DNA

<213> Canis familiaris

<220>

<221> CDS

<222> (66)..(689)

<400> 3

ctcatctgcc atgacacctt ccggtgggtg gggatgtgtg tgggtaaact ggcccactgg 60

gaacc atg gag tct gag gaa atc tac acg aat cag aag gtc gag atg cag 110

Met Glu Ser Glu Glu Ile Tyr Thr Asn Gln Lys Val Glu Met Gln

1 5 10 15

gca gcc ttc aaa gac aag aaa cag agg gtc cca gct gat aag gaa ggt 158

Ala Ala Phe Lys Asp Lys Lys Gln Arg Val Pro Ala Asp Lys Glu Gly

20 25 30

gca gat aac cct gac tat gag aat atc acc ttg gcc ttc aga aac cag 206

Ala Asp Asn Pro Asp Tyr Glu Asn Ile Thr Leu Ala Phe Arg Asn Gln

35 40 45

gac cag cca aag ggc agc cat tta cca ccc aag aat cag agc aag cag 254

Asp Gln Pro Lys Gly Ser His Leu Pro Pro Lys Asn Gln Ser Lys Gln

50 55 60

cca cct gcc agg aca cat cac acg gcc ttg gga ggg gcc cac gtc cca 302

Pro Pro Ala Arg Thr His His Thr Ala Leu Gly Gly Ala His Val Pro

65 70 75

acc ctg tct agg ctg ccc tca gac tct ggc cag ctc ccc cgt tgt ctg 350

Thr Leu Ser Arg Leu Pro Ser Asp Ser Gly Gln Leu Pro Arg Cys Leu

80 85 90 95

cac aga gtc atc atg agc ctg tac atg ctc ctc gcc ctg tcc tgc atc 398

His Arg Val Ile Met Ser Leu Tyr Met Leu Leu Ala Leu Ser Cys Ile

100 105 110

att ctc tta gtc ttg gtc ctc atg aag aat ctg gag atg tcc cag gag 446

Ile Leu Leu Val Leu Val Leu Met Lys Asn Leu Glu Met Ser Gln Glu

115 120 125

ttg ctg gcc ctg aaa agg gag ctc tgg aat gtg tcc gtc tcg gtg caa 494

Leu Leu Ala Leu Lys Arg Glu Leu Trp Asn Val Ser Val Ser Val Gln

130 135 140

gag tgc cag gag cag cag aat cag ggc tgg agc acc gtc cgg cag ctc 542

Glu Cys Gln Glu Gln Gln Asn Gln Gly Trp Ser Thr Val Arg Gln Leu

145 150 155

ctg gtg gag gcc aag cgt gac att tcc atg gtc ggg aga aat gcc cag 590

Leu Val Glu Ala Lys Arg Asp Ile Ser Met Val Gly Arg Asn Ala Gln

160 165 170 175

ctt gcg agt gag aag gtg aag acg ctg aca gca gac ata agc cat atc 638

Leu Ala Ser Glu Lys Val Lys Thr Leu Thr Ala Asp Ile Ser His Ile

180 185 190

aag agt aag tta cag gaa atc tcc aag atg ctg gag aag cca aag cca 686

Lys Ser Lys Leu Gln Glu Ile Ser Lys Met Leu Glu Lys Pro Lys Pro

195 200 205

tag acctcaacat acgcgaggac atcgaagccc tggctgcagc ttggcggacg 739

gggctgcgcc tcccagtgaa gatggccacg tgtgtgcacc acgtgtgttg tgagcctaag 799

gcgtgacaca gtgggtggct gtgtcagcag ggaccacgaa agtgtgtcag cgtgttgttg 859

gcagcatgtg tagcaccgtg aacgcgtgtg actgtcctgt ggtatgttgt gtgtaaatgt 919

gtcacggcag agccgtggcg ggggcacccc acgtgtcact gtaattgtgg gtgccctgtc 979

actacctgtg ttggtgtgaa caggtgtctg ccaacgagcg actgaggatg tcacggaggg 1039

ggttcggagc atgtacacat gtatgtccat ttgttcccgc gctcacgttg tgtgatttgt 1099

gaagataaag gccgatggaa aagaa 1124

<210> 4

<211> 207

<212> PRT

<213> Canis familiaris

<400> 4

Met Glu Ser Glu Glu Ile Tyr Thr Asn Gln Lys Val Glu Met Gln Ala

1 5 10 15

Ala Phe Lys Asp Lys Lys Gln Arg Val Pro Ala Asp Lys Glu Gly Ala

20 25 30

Asp Asn Pro Asp Tyr Glu Asn Ile Thr Leu Ala Phe Arg Asn Gln Asp

35 40 45

Gln Pro Lys Gly Ser His Leu Pro Pro Lys Asn Gln Ser Lys Gln Pro

50 55 60

Pro Ala Arg Thr His His Thr Ala Leu Gly Gly Ala His Val Pro Thr

65 70 75 80

Leu Ser Arg Leu Pro Ser Asp Ser Gly Gln Leu Pro Arg Cys Leu His

85 90 95

Arg Val Ile Met Ser Leu Tyr Met Leu Leu Ala Leu Ser Cys Ile Ile

100 105 110

Leu Leu Val Leu Val Leu Met Lys Asn Leu Glu Met Ser Gln Glu Leu

115 120 125

Leu Ala Leu Lys Arg Glu Leu Trp Asn Val Ser Val Ser Val Gln Glu

130 135 140

Cys Gln Glu Gln Gln Asn Gln Gly Trp Ser Thr Val Arg Gln Leu Leu

145 150 155 160

Val Glu Ala Lys Arg Asp Ile Ser Met Val Gly Arg Asn Ala Gln Leu

165 170 175

Ala Ser Glu Lys Val Lys Thr Leu Thr Ala Asp Ile Ser His Ile Lys

180 185 190

Ser Lys Leu Gln Glu Ile Ser Lys Met Leu Glu Lys Pro Lys Pro

195 200 205

<210> 5

<211> 1416

<212> DNA

<213> Felis catus

<220>

<221> CDS

<222> (24)..(641)

<400> 5

tctgcttgaa tcaggaggtc agg atg caa gca gca gac ttc aaa ggc aag aaa 53

Met Gln Ala Ala Asp Phe Lys Gly Lys Lys

1 5 10

cag agg gcc cca gac cat aag gaa ggt tcg gta cct caa ggt gca gac 101

Gln Arg Ala Pro Asp His Lys Glu Gly Ser Val Pro Gln Gly Ala Asp

15 20 25

cct gac tat gag aat atc acc ttg acc ttc aga aac cag gag caa cca 149

Pro Asp Tyr Glu Asn Ile Thr Leu Thr Phe Arg Asn Gln Glu Gln Pro

30 35 40

agg ggc agc cat tca cca ccc aag aat cga ggc aag cag cca cct gcc 197

Arg Gly Ser His Ser Pro Pro Lys Asn Arg Gly Lys Gln Pro Pro Ala

45 50 55

agc ccg cac ctc aca gcc tcg gga ggg gcc cct gtc cca gcc tgg tcg 245

Ser Pro His Leu Thr Ala Ser Gly Gly Ala Pro Val Pro Ala Trp Ser

60 65 70

aag cag gcc cca gac tct gcc cag gtc cct cgt tgg ctg cac aga gtc 293

Lys Gln Ala Pro Asp Ser Ala Gln Val Pro Arg Trp Leu His Arg Val

75 80 85 90

acc ctg agc ctg tac atc ctc ctt gcc ctg ttc tgc atc gtt ctc ttg 341

Thr Leu Ser Leu Tyr Ile Leu Leu Ala Leu Phe Cys Ile Val Leu Leu

95 100 105

gcc ttg gtc ctg gtg aag aat tct gag gtg tcc cag gag ctg ctg gtc 389

Ala Leu Val Leu Val Lys Asn Ser Glu Val Ser Gln Glu Leu Leu Val

110 115 120

gtg aaa agg gag ctc cag aat gtc tcc atc tcg gga caa cag tgt cag 437

Val Lys Arg Glu Leu Gln Asn Val Ser Ile Ser Gly Gln Gln Cys Gln

125 130 135

gag gag cag aaa cag ggc tgg agc agc gtc cag cag ctc atc acg gag 485

Glu Glu Gln Lys Gln Gly Trp Ser Ser Val Gln Gln Leu Ile Thr Glu

140 145 150

gcc agg cag gac att gac atg atc aag aga aat gtc cac atc ggg aac 533

Ala Arg Gln Asp Ile Asp Met Ile Lys Arg Asn Val His Ile Gly Asn

155 160 165 170

gag aaa gtg aag acg ctg tca aca gac tta agc caa atc aag act aaa 581

Glu Lys Val Lys Thr Leu Ser Thr Asp Leu Ser Gln Ile Lys Thr Lys

175 180 185

tta cat gaa atc tcc aag ata cta gag aag aag ccg cag cca cag ccc 629

Leu His Glu Ile Ser Lys Ile Leu Glu Lys Lys Pro Gln Pro Gln Pro

190 195 200

aca gct caa taa atgagaagac attgacaccc aggctgcagc ttggaggacg 681

Thr Ala Gln

205

gggctgcact tccccgtgaa gacggccgca tgtgtgcctc atggtgtcac gggagcgata 741

acacatgata cagtgggcgg ctgtgtcagc aaggaccgca gaagtgtgtc agcctgggcg 801

861 cgggtgttgg taacacgtgt tgcactgtga

tgtgaatgtc atggagctgt gtgtgtgtgt gcgcgtgcgt gtgtgtgtgt ggagcacccc 921

ctacatcact gtaactgcgg gtgttgaggg tgtaccccgt cactccctgt gttgtgtgaa 981

caggtgggtg tcagtgtgtg actgattatg tcaccgaggg tgtgcagagc gggtacattt 1041

gtgcgttccc ttgtttctgc actcacgttt tgtgatttgt gacgataaag gccaatggga 1101

aagaatgtgg ctttcagatc tgttcctggg agcatctgggg ggtgggggtg gggacccggt 1161

ggcggagggt ctgcaagtat taagggatga ggaaagtcac acagcaagca cgcggacgtg 1221

ataaccagga gccctggggg cacgagtgtg tgtgagcatg aatgccctga atgggtcctt 1281

ttgtgcccat gaacttgtac ccagcaagga acagtctccg tgtctgagac tgtgtgccca 1341

gcagggtttg tggcccgaga tatacactgt ttccctaagt ggggctcctg ggtggctcag 1401

tcggttaagc gtccg 1416

<210> 6

<211> 205

<212> PRT

<213> Felis catus

<400> 6

Met Gln Ala Ala Asp Phe Lys Gly Lys Lys Gln Arg Ala Pro Asp His

1 5 10 15

Lys Glu Gly Ser Val Pro Gln Gly Ala Asp Pro Asp Tyr Glu Asn Ile

20 25 30

Thr Leu Thr Phe Arg Asn Gln Glu Gln Pro Arg Gly Ser His Ser Pro

35 40 45

Pro Lys Asn Arg Gly Lys Gln Pro Pro Ala Ser Pro His Leu Thr Ala

50 55 60

Ser Gly Gly Ala Pro Val Pro Ala Trp Ser Lys Gln Ala Pro Asp Ser

65 70 75 80

Ala Gln Val Pro Arg Trp Leu His Arg Val Thr Leu Ser Leu Tyr Ile

85 90 95

Leu Leu Ala Leu Phe Cys Ile Val Leu Leu Ala Leu Val Leu Val Lys

100 105 110

Asn Ser Glu Val Ser Gln Glu Leu Leu Val Val Lys Arg Glu Leu Gln

115 120 125

Asn Val Ser Ile Ser Gly Gln Gln Cys Gln Glu Glu Gln Lys Gln Gly

130 135 140

Trp Ser Ser Val Gln Gln Leu Ile Thr Glu Ala Arg Gln Asp Ile Asp

145 150 155 160

Met Ile Lys Arg Asn Val His Ile Gly Asn Glu Lys Val Lys Thr Leu

165 170 175

Ser Thr Asp Leu Ser Gln Ile Lys Thr Lys Leu His Glu Ile Ser Lys

180 185 190

Ile Leu Glu Lys Lys Pro Gln Pro Gln Pro Thr Ala Gln

195 200 205

<210> 7

<211> 1132

<212> DNA

<213> Mus musculus

<220>

<221> CDS

<222> (21)..(572)

<400> 7

acgtgaatca accaagcaga atg cat gca tca gcc tcc cag gat aag aac cgg 53

Met His Ala Ser Ala Ser Gln Asp Lys Asn Arg

1 5 10

agg aag cca ggt cat gat gaa ggt gct cac aat cct gac tac gag aat 101

Arg Lys Pro Gly His Asp Glu Gly Ala His Asn Pro Asp Tyr Glu Asn

15 20 25

ata acc ttg gcc ttc aga aac aag gac caa ctc aaa ctc agc caa tca 149

Ile Thr Leu Ala Phe Arg Asn Lys Asp Gln Leu Lys Leu Ser Gln Ser

30 35 40

aca ccc aca aaa caa gcc aag ttc aag aca tcc ctg gac cca gct gag 197

Thr Pro Thr Lys Gln Ala Lys Phe Lys Thr Ser Leu Asp Pro Ala Glu

45 50 55

tcc ccg cct tgg ttg tac aga acc att atg atg ttg tat gtt ctc ctt 245

Ser Pro Pro Trp Leu Tyr Arg Thr Ile Met Met Leu Tyr Val Leu Leu

60 65 70 75

gct ctc gtc ttt tta tcc tgc atc gtc ctc tct gct ttg gtc ttg gtg 293

Ala Leu Val Phe Leu Ser Cys Ile Val Leu Ser Ala Leu Val Leu Val

80 85 90

aaa aat tct gag atg tcc aag gag ctg tgg acc ttg aaa gca gag ctt 341

Lys Asn Ser Glu Met Ser Lys Glu Leu Trp Thr Leu Lys Ala Glu Leu

95 100 105

tcg aat gtt tca gac acg gtg tgg aat atc cgg gag ctc cag aat cag 389

Ser Asn Val Ser Asp Thr Val Trp Asn Ile Arg Glu Leu Gln Asn Gln

110 115 120

caa acg agg att tgg gaa gct gcc cag ggg gac atc aag gag gtc aag 437

Gln Thr Arg Ile Trp Glu Ala Ala Gln Gly Asp Ile Lys Glu Val Lys

125 130 135

aag acc ctt ggc aca gtc atg agt agc atc cag act gga aac gac cgg 485

Lys Thr Leu Gly Thr Val Met Ser Ser Ile Gln Thr Gly Asn Asp Arg

140 145 150 155

ctg aag act gtg ccg gca gat ata acc caa atc aag aaa act ctt gag 533

Leu Lys Thr Val Pro Ala Asp Ile Thr Gln Ile Lys Lys Thr Leu Glu

160 165 170

gcg cta gaa aag aag gca cag cct cag ccc agt aca taa gaggacacca 582

Ala Leu Glu Lys Lys Ala Gln Pro Gln Pro Ser Thr

175 180

cagcagtacc tgtgaagact ccgaattgca cctgctagtg aagatggcag atgggggtgg 642

gtactgagct tgagtgtgaa cctgccgtgc atcctcatat aaaaaagatt ctccaccagg 702

gggaatgagt gttgaagagg tgtgtatgca aatgagcatt tggggtttcc atgtattcca 762

ggagaagggt ttatggtgga aagagaacat ggcagtcaca gcaggtgtta ctctttatgg 822

gccacatagg tgtatgccct ggcttatgtg agtataggca tgtcctggtt ggcagctatt 882

cccgagaagt ccccaaagtg taagtgacat gtaggacatg cctccccatt ctcttgctca 942

tgtatgtgca tctggctgtt ctgtatgtgt gtcactgaag tggtgggtga tagacatcac 1002

cctggagatg tgtcatggca tgggtcattc ctagtgtttt tggtcatgtc agcttgtgtg 1062

ttcagggcat gcacacaaat gtagccatcg atttctgcac ttgtatttat gattcaagaa 1122

gataaatgcc 1132

<210> 8

<211> 183

<212> PRT

<213> Mus musculus

<400> 8

Met His Ala Ser Ala Ser Gln Asp Lys Asn Arg Arg Lys Pro Gly His

1 5 10 15

Asp Glu Gly Ala His Asn Pro Asp Tyr Glu Asn Ile Thr Leu Ala Phe

20 25 30

Arg Asn Lys Asp Gln Leu Lys Leu Ser Gln Ser Thr Pro Thr Lys Gln

35 40 45

Ala Lys Phe Lys Thr Ser Leu Asp Pro Ala Glu Ser Pro Pro Trp Leu

50 55 60

Tyr Arg Thr Ile Met Met Leu Tyr Val Leu Leu Ala Leu Val Phe Leu

65 70 75 80

Ser Cys Ile Val Leu Ser Ala Leu Val Leu Val Lys Asn Ser Glu Met

85 90 95

Ser Lys Glu Leu Trp Thr Leu Lys Ala Glu Leu Ser Asn Val Ser Asp

100 105 110

Thr Val Trp Asn Ile Arg Glu Leu Gln Asn Gln Gln Thr Arg Ile Trp

115 120 125

Glu Ala Ala Gln Gly Asp Ile Lys Glu Val Lys Lys Thr Leu Gly Thr

130 135 140

Val Met Ser Ser Ile Gln Thr Gly Asn Asp Arg Leu Lys Thr Val Pro

145 150 155 160

Ala Asp Ile Thr Gln Ile Lys Lys Thr Leu Glu Ala Leu Glu Lys Lys

165 170 175

Ala Gln Pro Gln Pro Ser Thr

180

<210> 9

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Primer T3

<400> 9

aattaaccct cactaaaggg 20

<210> 10

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Primer T7

<400> 10

taatacgact cactatagg 19

<210> 11

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Sense primer for canine RT

<400> 11

ccacgtccca accctgtcta 20

<210> 12

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Sense primer for canine RT

<400> 12

gtgctccagc cctgattctg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Sense primer for human RT

<400> 13

tggccttcaa aaatcaggac 20

<210> 14

<211> 21

<212> DNA

<213> Artificial

<220>

<223> Human RT antisense primer

<400> 14

aggctcagga tggctctgta c 21

<210> 15

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Sense primer for mouse RT

<400> 15

tccaaggagc tgtggacctt 20

<210> 16

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Mouse RT antisense primer

<400> 16

agtcttcagc cggtcgtttc 20

<210> 17

<211> 37

<212> DNA

<213> Artificial

<220>

<223> Murine mcemp1 EcoRI sense primer

<400> 17

gaattcgccg ccaccatgca tgcatcagcc tcccagg 37

<210> 18

<211> 29

<212> DNA

<213> Artificial

<220>

<223> Mouse mcemp1 antisense NotI primer

<400> 18

gcggccgctt atgtactgggg ctgaggctg 29

<210> 19

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Human MCEMP1 EcoRI sense primer

<400> 19

gaattcgccg ccaccatgga agtggaggaa atctacaag 39

<210> 20

<211> 29

<212> DNA

<213> Artificial

<220>

<223> Human MCEMP1 antisense NotI primer

<400> 20

gcggccgctt attgaggtga ggactgtgg 29

<---

Claims (8)

1. An immunoinducing composition for use in a method for treating or preventing MCEMP1-expressing cancer in an individual in need thereof, comprising as an active ingredient an effective amount of at least one polypeptide having immunoinducing activity and consisting of the amino acid sequence shown in SEQ ID NO: 2 or 8 according to the sequence listing, or a recombinant vector containing a polynucleotide encoding a polypeptide and capable of expressing said polypeptide in vivo , and wherein the polypeptide is administered to an individual that is the same individual as the individual of the mammalian species from which the polypeptide was isolated. 2. The immunoinducing composition of claim 1, wherein the cancer is leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma, or perianal adenocarcinoma. 3. Immunoinducing composition according to any one of paragraphs. 1 or 2, also containing an immunostimulant or combined with an immunostimulant in the form of a separate preparation. 4. The immunostimulatory composition according to claim 3, where the immunostimulant is at least one agent selected from the group consisting of Freund's incomplete adjuvant, Montanide, Poly-IC and its derivatives, CpG oligonucleotides, interleukin 12, interleukin 18, interferon α, interferon β, interferon ω, interferon γ and Flt 3 ligand. 5. A method of treating or preventing MCEMP1-expressing cancer, comprising administering to an individual in need thereof at least one polypeptide having immunoinducing activity and consisting of the amino acid sequence represented by SEQ ID NO: 2 or 8 according to the sequence listing, or a recombinant vector, containing a polynucleotide encoding a polypeptide and capable of expressing said polypeptide in vivo , and wherein the polypeptide is administered to an individual that is the same individual as the individual of the mammalian species from which the polypeptide was isolated. 6. A method for treating or preventing an MCEMP1-expressing cancer, comprising administering to an individual in need thereof, (1) antigen-presenting cells treated with at least one polypeptide having immunoinducing activity and consisting of the amino acid sequence represented by SEQ ID NO: 2 or 8 according to the sequence listing, or a recombinant vector containing a polynucleotide encoding a polypeptide and capable of expressing said polypeptide in cells ; or (2) T cells treated with antigen-presenting cells according to the above subparagraph (1), where the individual is the same individual as the individual of the mammalian species from which the polypeptide was isolated.

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