Specification
Title of invention: immune inducing agent
Technical field
[0001]
　The present invention relates to a novel immunity inducing agent useful as a therapeutic and / or prophylactic agent for cancer and the like.
BACKGROUND ART
[0002]
　Cancer is the leading cause of all deaths, and the treatment currently being performed is a combination of radiotherapy and chemotherapy mainly based on surgical therapy. Despite the recent development of new surgical methods and the discovery of new anticancer drugs, the results of cancer treatment have not improved so much, except for some cancers. In recent years, with the progress of molecular biology and cancer immunology, cancer antigens recognized by cytotoxic T cells reactive to cancer, genes encoding cancer antigens, and the like have been identified, and expectation for antigen specific immunotherapy is high It is increasing.
[0003]
　Melanocortin 2 receptor accessory protein 2 (MRAP 2) is a type 1 or type 2 transmembrane protein, which is involved in the regulation of melanocortin receptor (MCR) activity and functions in in vivo energy metabolism (Non-Patent Document 1 ). It has also been reported that obesity occurs in MRAP 2 deficient mice despite the lack of overeating, and it has been reported that some patients with severe obesity also have mutations in MRAP 2 gene in humans (Non-Patent Document 2). However, there is no report that the MRAP2 protein has an immunity inducing activity against cancer cells, whereby the protein is useful for the treatment or prevention of cancer.
Prior Art Document
Non-patent literature
[0004]
Non-Patent Document 1: Jackson DS. Et al. Front. Neurosci, 9: 213 (2015)
Non-Patent Document 2: Asai M. et al. Science, 341: 275-278 (2013)
Summary of the invention
Problem to be Solved by Invention
[0005]
　An object of the present invention is to find a novel polypeptide useful as a therapeutic and / or prophylactic agent for cancer, and to provide use of the polypeptide as an immunity inducing agent.
Means for solving the problem
[0006]
　As a result of earnest research, the inventors of the present invention have found that cDNA encoding a protein that binds to an antibody present in serum derived from a cancer-bearing living body is obtained by a SEREX method using a canine testis-derived cDNA library and a serum of a leukemia dog Based on the cDNA, a polypeptide of canine Melanocortin 2 receptor accessory protein 2 (hereinafter referred to as MRAP 2) having the amino acid sequence represented by SEQ ID NO: 4 was prepared. In addition, human, feline and mouse MRAP2 polypeptides having the amino acid sequences represented by SEQ ID NO: 2, 6, and 8 were prepared based on the obtained human, feline and mouse homology genes of the canine gene. These MRAP2 polypeptides are expressed in leukemia, malignant lymphoma, lung cancer, brain tumor, colon cancer, melanoma, neuroblastoma, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma, and perianal adenocarcinoma I found out that. Furthermore, we found that by administering these MRAP 2 to the living body, it is possible to induce immune cells against MRAP 2 in the living body and regress tumors in vivo expressing MRAP 2. Furthermore, it was found that a recombinant vector capable of expressing a polynucleotide encoding a polypeptide of MRAP2 or a fragment thereof induces an antitumor effect in vivo against cancer expressing MRAP2.
[0007]
　Furthermore, it has been found that the polypeptide of MRAP2 is presented by antigen presenting cells and has the ability to activate and proliferate cytotoxic T cells specific for the polypeptide (also referred to as "immunity inducing activity"), Therefore, the polypeptide is useful for the treatment and / or prevention of cancer, and antigen-presenting cells contacted with the polypeptide and T cells contacted with the antigen-presenting cells are useful for treating and / or preventing cancer And found that the present invention was completed.
[0008]
　Accordingly, the present invention includes the following inventions.
(1) A polypeptide comprising at least one polypeptide selected from the following polypeptides (a) to (d) and having immunity-inducing activity, or a polynucleotide encoding the polypeptide, , As an active ingredient.
(A) a polypeptide consisting of at least 7 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing up to the full length of the polypeptide
(b) SEQ ID NO: 2, 4, 6 , Or a polypeptide consisting of an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid
sequence represented by SEQ ID NO: 2, 4, 6, or 8 in the sequence listing (c) A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the sequence
(d) a polypeptide comprising as a partial sequence any one of the above (a) to (c)
(2) The immunity inducing agent according to (1), which is a treating agent.
(3) The immunity-inducing agent according to (1), which is an active ingredient of a therapeutic and / or prophylactic agent for cancer.
(4) The immunity inducing agent according to (3), wherein the cancer is a cancer expressing MRAP2.
(5) The method according to (1), wherein the cancer is leukemia, malignant lymphoma, lung cancer, brain tumor, colon cancer, melanoma, neuroblastoma, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma or perianal adenocarcinoma (3) or (4) above.
(6) The immunity inducing agent according to any one of (1) to (5), further comprising an immunopotentiating agent.
(7) The method according to any one of (1) to (4), wherein the immunopotentiator is Freund's incomplete adjuvant, Montanide, poly IC and its derivatives, CpG oligonucleotide, Interleukin 12, Interleukin 18, Interferon α, Interferon β, Interferon ω, Interferon γ and Flt 3 Ligand (6), wherein the immunity-inducing agent is at least one member selected from the group consisting of:
(8) A method for producing an antigen-presenting cell comprising a complex of the polypeptide and MHC molecule, comprising bringing a polypeptide defined in (1) and an antigen-presenting cell derived from a subject into contact with each other ex vivo or in vitro .
(9) The method according to (8), wherein the antigen presenting cells are dendritic cells or B cells carrying MHC class I molecules.
(10) contacting an antigen-presenting cell obtained by the method according to (8) or (9) with a T cell derived from a subject ex vivo or in vitro to activate the T cell. A method for producing cytotoxic T cells specific to a polypeptide defined in (a).
(11) A pharmaceutical composition comprising at least one polypeptide selected from the following polypeptides (a) to (d) and having immunity-inducing activity, or a polynucleotide encoding the polypeptide, And a recombinant vector capable of expressing the polypeptide of the present invention to a subject.
(A) a polypeptide consisting of at least 7 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing up to the full length of the polypeptide
(b) SEQ ID NO: 2, 4, 6 , Or a polypeptide consisting of an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:
(C) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in Sequence Listing
(d) The
　present application claims priority to Japanese Patent Application No. 2016-064032 filed on Mar. 28, 2016, and the specification of the patent application Including the contents described in.
Effect of the invention
[0009]
　INDUSTRIAL APPLICABILITY The present invention provides a novel immunity inducing agent useful for the treatment and / or prevention of cancer. When the polypeptide used in the present invention is administered to a subject, as specifically shown in the examples described later, it is possible to induce immune cells in vivo and to further reduce or regress existing cancer . Accordingly, the polypeptide is useful for treating or preventing cancer.
Brief Description of the Drawings
[0010]
FIG. 1 is a diagram showing an expression pattern of the identified MRAP2 gene in a tumor tissue of a dog. Reference number 1; shows the expression pattern of the canine MRAP2 gene in each of the tumor tissues of the dogs shown.
FIG. 2 shows the expression pattern of the identified MRAP2 gene in each human tissue and cancer cell line. Reference number 2: shows the expression pattern of human MRAP2 gene in each human normal human tissue shown. Reference number 3 shows the expression pattern of human MRAP2 gene in each human cancer cell line shown in the figure. In the figure, PBMC represents peripheral blood mononuclear cells.
FIG. 3 is a diagram showing the expression pattern of the identified MRAP2 gene in each mouse cancer cell line. Reference number 4 shows the expression pattern of mouse MRAP2 gene in each cancer cell line of the mouse shown.
MODE FOR CARRYING OUT THE INVENTION
[0011]
1. Polypeptides Examples of polypeptides
　contained as an active ingredient in the immunity inducing agent of the present invention include the following polypeptides (a) to (d). In the present invention, the term "polypeptide" refers to a molecule formed by binding a plurality of amino acids with a peptide, and includes not only a polypeptide molecule having a large number of amino acids constituting it, but also a molecule of a low molecular weight having a small number of amino acids (Oligopeptide) and full-length protein are also included.
(A) a polypeptide comprising an amino acid sequence of at least 7 consecutive amino acids in the polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8 in the Sequence Listing and having an immunity inducing activity
(b ) A polypeptide consisting of an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 in the Sequence Listing and which has immunity inducing activity
(c ) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 of the sequence listing and having immunity inducing activity
(d) ) To (c) as a partial sequence and having immunity inducing activity
[0012]
　In the present invention, "having an amino acid sequence" means that amino acid residues are arranged in such order. Thus, for example, "a polypeptide having the amino acid sequence shown in SEQ ID NO: 8" refers to a polypeptide having the amino acid sequence shown in SEQ ID NO: 8, Met, Glu, Met, Ser, Ala ...... Ile, Asp, Leu, Asp And having a size of 207 amino acid residues. Further, for example, "the polypeptide having the amino acid sequence represented by SEQ ID NO: 8" may be abbreviated as "the polypeptide of SEQ ID NO: 8" in some cases. This also applies to the expression "having a base sequence". In this case, the term "having" may be replaced with the expression "consisting of".
[0013]
　Here, "immunity inducing activity" means the ability to induce immune cells secreting cytokines such as interferon in vivo.
[0014]
　Whether or not the polypeptide has an immunity inducing activity can be confirmed using, for example, a well-known ELISPOT assay or the like. Specifically, cells such as peripheral blood mononuclear cells are obtained from a living body to which a polypeptide to be evaluated for immunity induction activity is administered, the cells are co-cultured with the polypeptide, and the amount of cytokine production from the cells , The number of immune cells in the cell can be measured by measuring with a specific antibody, so that the immunity inducing activity can be evaluated.
[0015]
　In addition, as described in Examples described later, when the recombinant polypeptides of (a) to (d) are administered to a cancer-bearing living body, the tumor can also be regressed by the immunity inducing activity thereof. Therefore, the immunity-inducing activity can also be evaluated as an ability to inhibit proliferation of cancer cells or reduce or eliminate cancer tissue (tumor) (hereinafter referred to as "antitumor activity"). The anti-tumor activity of the polypeptide is confirmed by actually examining whether the tumor is shrunk or the like by actually administering the polypeptide to the cancer-bearing living body as specifically described in Examples described later can do. It is also possible to evaluate the antitumor activity of a polypeptide by examining whether cytotoxic T cells induced by administering the polypeptide to a cancer-bearing living body show cytotoxic activity against a tumor it can. Measurement of cytotoxic activity of T cells in vivo can be confirmed by investigating whether the tumor is increased or the like by, for example, administering an antibody that removes the T cell from the living body but limited to this It is not done.
[0016]
　Alternatively, it is necessary to investigate whether T cells stimulated with the polypeptide (that is, T cells contacted with antigen presenting cells presenting the polypeptide) show cytotoxic activity against tumor cells in vitro , The antitumor activity of the polypeptide can also be evaluated. The contact between the T cell and the antigen presenting cell can be carried out by co-culturing both the T cell and the antigen presenting cell in a liquid medium as described later. Measurement of the cytotoxic activity can be carried out by a known method called 51 Cr release assay described, for example, in Int. J. Cancer, 58: p 317, 1994 . When the polypeptide is used for cancer treatment and / or prophylaxis, there is no particular limitation, but it is preferable to evaluate the immunity induction activity using antitumor activity as an index.
[0017]
　The amino acid sequences shown in SEQ ID NOS: 2, 4, 6, or 8 in the Sequence Listing disclosed by the present invention were determined by SEREX method using a canine testis-derived cDNA library and a serum of a cancer-bearing dog, It is the amino acid sequence of MRAP2 isolated as a polypeptide that binds to antibodies specifically present in the serum and its human, cat, and mouse homologous factor (see Example 1). In human MRAP2 which is a human homologous factor of canine MRAP2, the sequence identity is 91% of the base sequence and 94% of the amino acid sequence, and in the feline homologous factor feline MRAP 2 the sequence identity is 95% of the base sequence and 96% of the amino acid sequence In mouse MRAP2 which is a mouse homologous factor, the sequence identity is 84% ​​for the base sequence and 88% for the amino acid sequence.
[0018]
　The polypeptide of the above (a) is a polypeptide consisting of 7 or more consecutive amino acids in the polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8, preferably from consecutive 8, 9 or 10 or more consecutive amino acids And has immunity inducing activity. Particularly preferably, the polypeptide has the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8. As is well known in this field, polypeptides of about 7 amino acid residues or more can exert antigenicity and immunogenicity. Therefore, as long as it is a polypeptide consisting of amino acids of from 7 consecutive amino acid residues (7) or more to all the amino acid residues (full length) in the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, immunity- , It can be used for the preparation of the immunity-inducing agent of the present invention.
[0019]
　In addition, as a principle of immunity induction by administering a cancer antigen polypeptide, a polypeptide is taken up by an antigen presenting cell and then degraded by peptidase in the cell to become a smaller fragment and presented on the surface of the cell It is known that it is recognized by cytotoxic T cells and the like, and selectively kills cells presenting the antigen. The size of the polypeptide presented on the surface of the antigen presenting cell is relatively small, about 7 to 30 amino acids. Accordingly, from the viewpoint of presenting on antigen presenting cells, it is preferable that the polypeptide of (a) is about 7 to 30 consecutive in the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 Is one of the preferred embodiments, and it is sufficient if it consists of about 8 to 30 or about 9 to 30 amino acids. These relatively small sized polypeptides may be directly displayed on the cell surface on the antigen presenting cells without being taken into the antigen presenting cells.
[0020]
　In addition, polypeptides incorporated into antigen-presenting cells undergo cleavage at random positions by peptidases within the cells to produce various polypeptide fragments, these polypeptide fragments are presented on the antigen presenting cell surface Therefore, if a large-sized polypeptide such as the full-length region of SEQ ID NO: 2, 4, 6, or 8 is administered, the polypeptide which is effective for immunity induction via antigen presenting cells by degradation in antigen presenting cells Fragments inevitably occur. Therefore, for immunity induction via antigen-presenting cells, a large-sized polypeptide can be preferably used, and the number of amino acids is preferably 30 or more, more preferably 100 or more, further preferably 200 or more, still more preferably SEQ ID NO: 2, 4 , 6, or 8, respectively.
[0021]
　The above-mentioned polypeptide (c) is a polypeptide in which a few (preferably one or several) amino acid residues of the polypeptide of the above (a) are substituted, deleted and / or inserted , Having a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more or 99.5% or more with the original sequence and having immunity inducing activity . Generally, in a protein antigen, even when a small number of amino acid residues in the amino acid sequence of the protein are substituted and deleted or inserted, the protein antigen may have almost the same antigenicity as the original protein Something is widely known in the art. Therefore, the polypeptide of the above (c) can also exhibit an immunity inducing activity, so it can be used for preparation of the immunity inducing agent of the present invention. In addition, the polypeptide of (b) above may have one to several amino acid residues among the amino acid sequences shown in SEQ ID NO: 2, 4, 6, or 8 substituted, deleted and / or inserted It is also preferred that it is a polypeptide. The term "several" in the present specification means an integer of 2 to 10, preferably an integer of 2 to 6, and more preferably an integer of 2 to 4.
[0022]
　Here, "sequence identity" of an amino acid sequence or base sequence means that both amino acid sequences (or bases) are aligned so that the amino acid residues (or bases) of two amino acid sequences (or base sequences) And aligning the number of identical amino acid residues (or the number of matched bases) by the total number of amino acid residues (or the total number of bases), in percentage. At the time of the above alignment, a gap is appropriately inserted into one or both of the two sequences to be compared, if necessary. The alignment of such sequences can be carried out using well-known algorithms such as BLAST, FASTA, CLUSTALW, for example. When a gap is inserted, the total number of amino acid residues is the number of residues in which one gap is counted as one amino acid residue. If the total number of amino acid residues counted in this manner is different between the two sequences to be compared, the sequence identity (%) is the total number of amino acid residues of the longer sequence, and the number of identical amino acid residues It is calculated by dividing the number.
[0023]
　The 20 kinds of amino acids constituting the natural protein are neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro) having a low polar side chain, neutral amino acids having a hydrophilic side chain (Asn Those having similar properties such as acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp), Gln, Thr, Ser, Tyr, Cys) , And it is known that the properties of polypeptides often do not change if they are substituted among them. Therefore, in the case of substituting the amino acid residues in the polypeptide of the above (a) of the present invention, substitution between each of these groups is preferable because it increases the possibility of maintaining the immunity inducing activity .
[0024]
　The above polypeptide (d) is a polypeptide which contains the polypeptide of any one of the above (a) to (c) as a partial sequence and has an immunity inducing activity. That is, it is a polypeptide obtained by adding another amino acid or polypeptide to one end or both ends of the polypeptide of any one of (a) to (c), and has immunity inducing activity. Such a polypeptide can also be used for the preparation of the immunity-inducing agent of the present invention.
[0025]
　The polypeptide can be synthesized according to a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method), or the like. It can also be synthesized by a conventional method using various kinds of commercially available peptide synthesizers. In addition, it is also possible to prepare a polynucleotide encoding the polypeptide using a known genetic engineering technique, introduce the polynucleotide into an expression vector, introduce the polynucleotide into a host cell, and produce the polypeptide in the host cell , A desired polypeptide can be obtained.
[0026]
　上記ポリペプチドをコードするポリヌクレオチドは、公知の遺伝子工学的手法や市販の核酸合成機を用いた常法により、容易に調製することができる。例えば、配列番号３の塩基配列を有するＤＮＡは、イヌ染色体ＤＮＡ又はｃＤＮＡライブラリーを鋳型として使用し、配列番号３に記載した塩基配列を増幅できるように設計した一対のプライマーを用いてＰＣＲを行うことにより調製することができる。配列番号１の塩基配列を有するＤＮＡであれば、上記鋳型としてヒト染色体ＤＮＡ又はｃＤＮＡライブラリーを使用することで同様に調製できる。ＰＣＲの反応条件は適宜設定することができ、例えば、９４℃で３０秒間（変性）、５５℃で３０秒～１分間（アニーリング）、７２℃で１分間（伸長）からなる反応工程を１サイクルとして、例えば３０サイクル行った後、７２℃で７分間反応させる条件などを挙げることができるが、これに限定されない。また、本明細書中の配列表の配列番号１、３に示される塩基配列及びアミノ酸配列の情報に基づいて、適当なプローブやプライマーを調製し、それを用いてヒトやイヌなどのｃＤＮＡライブラリーをスクリーニングすることにより、所望のＤＮＡを単離することができる。ｃＤＮＡライブラリーは、配列番号２、４のタンパク質を発現している細胞、器官又は組織から作製することが好ましい。上記のプローブ又はプライマーの調製、ｃＤＮＡライブラリーの構築、ｃＤＮＡライブラリーのスクリーニング、ならびに目的遺伝子のクローニングなどの操作は当業者に既知であり、例えば、モレキュラークローニング第２版、カレント・プロトコールズ・イン・モレキュラバイオロジー等に記載された方法に準じて行うことができる。このようにして得られたＤＮＡから、上記（ａ）のポリペプチドをコードするＤＮＡを得ることができる。また、各アミノ酸をコードするコドンは公知であるから、特定のアミノ酸配列をコードするポリヌクレオチドの塩基配列は容易に特定することができる。従って、上記の（ｂ）～（ｄ）のポリペプチドをコードするポリヌクレオチドの塩基配列も容易に特定することができるので、そのようなポリヌクレオチドも、市販の核酸合成機を用いて常法により容易に合成することができる。
[0027]
　The host cell may be any cell as long as it is capable of expressing the polypeptide. Examples of prokaryotic cells include Escherichia coli and examples of eukaryotic cells include monkey kidney cells COS 1, Chinese hamster ovary cells Mammalian cultured cells such as CHO, budding yeast, fission yeast, silkworm cell, Xenopus egg cell and the like, but it is not limited thereto.
[0028]
　When prokaryotic cells are used as host cells, expression vectors having origins, promoters, ribosome binding sites, DNA cloning sites, terminators and the like capable of replication in prokaryotic cells are used as expression vectors. Examples of expression vectors for E. coli include pUC system, pBluescript II, pET expression system, pGEX expression system and the like. By incorporating the DNA encoding the polypeptide into such an expression vector, transforming the prokaryotic host cell with the vector, and culturing the obtained transformant, the polypeptide encoded by the DNA can be transformed into a prokaryotic Can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.
[0029]
　When eukaryotic cells are used as host cells, expression vectors for eukaryotic cells having promoters, splicing regions, poly (A) addition sites, etc. are used as expression vectors. Examples of such expression vectors are exemplified by pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag (A, B, C), pMSG, pYES2 it can. Similarly to the above, if the DNA encoding the polypeptide is incorporated into such an expression vector, the eukaryotic host cell is transformed with the vector, and the resultant transformant is cultured, the DNA encodes Can be expressed in a eukaryotic host cell. In the case of using pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like as an expression vector, fusion with addition of various tags such as His tag, FLAG tag, myc tag, HA tag, As the protein, the polypeptide can be expressed.
[0030]
　For introduction of the expression vector into the host cell, well-known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method and the like can be used.
[0031]
　In order to isolate and purify a polypeptide of interest from a host cell, known isolation procedures can be used in combination. For example, treatment with a denaturing agent such as urea or surfactant, ultrasonic treatment, enzyme digestion, salting out and solvent fraction precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, But are not limited to, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.
[0032]
　The polypeptide obtained by the above method includes those which are in the form of a fusion protein with any other protein, as described above. For example, a fusion protein with glutathione-S-transferase (GST) or a His tag can be exemplified. Polypeptides in the form of such fusion proteins are also included in the scope of the present invention as the polypeptide of (d) above. Furthermore, the polypeptide expressed in the transformed cell may undergo various modifications within the cell after being translated. Such a post-translationally modified polypeptide also falls within the scope of the present invention as long as it has immunity inducing activity. Examples of such translational modifications include elimination of N-terminal methionine, N-terminal acetylation, glycosylation, limited degradation by intracellular proteases, myristoylation, isoprenylation, phosphorylation and the like.
[0033]
2. Immunogene inducing agent As
　specifically described in the Examples described later, when a polypeptide having the immunity-inducing activity is administered to a cancer-bearing living body, the tumor can be regressed. Therefore, the immunity-inducing agent of the present invention can be used as a therapeutic and / or prophylactic agent for cancer. Furthermore, the polypeptide having immunity-inducing activity can be used for the treatment and / or prophylaxis of cancer by immunity induction.
[0034]
　Here, the terms "tumor" and "cancer" mean malignant neoplasms and are used interchangeably.
[0035]
　In this case, the cancer to be treated is not particularly limited as long as it is a cancer that expresses MRAP2, but is preferably a cancer that expresses MRAP2 significantly more than normal cells, specifically, leukemia, malignant Lymphoma, lung cancer, brain tumor, colon cancer, melanoma, neuroblastoma, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma or perianal adenocarcinoma. These specific cancers include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T cell leukemia, aleukocytic Leukemia, leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonic cellular leukemia, eosinophilic leukemia, gross leukemia, leader Cellular leukemia, Schilling leukemia, stem cell leukemia, subleukaemic leukemia, undifferentiated cellular leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, tissue Spherical leukemia, stem cell leukemia, acute monocytic leukemia, leukopenia Lymphocytic leukemia, lymphoblastic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphotrophic leukemia, lymphoid leukemia, lymphosarcoma cellular leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, simple Bone marrow lymphocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, neagle leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, non-Hodgkin's lymphoma (bar Lymphoma of the kidney (BL), small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLCL), marginal zone Marginal zone lymphoma (MZL), hairy cell leukemia (HCL), lymphoplasmacytic leukemia (LPL), mucosa-associated lymph Woven extranodal marginal zone B cell lymphoma (MALT), mediastinal large cell lymphoma, intravascular large Primary T cell and NK cell lymphoma (precursor T cell lymphoblastic lymphoma, NK blast cell lymphoma), tumors of mature T and NK cells, cell lymphoma, primary effusion lymphoma, precursor B cell lymphoblastic leukemia / lymphoma, (Including peripheral T cell lymphoma and leukemia (PTL)), adult T cell leukemia / T cell lymphoma and large granular lymphocytic leukemia, T cell chronic lymphocytic leukemia / prolymphocytic leukemia, T cell large granule Lymphocytic leukemia, aggressive NK cell leukemia, extra-nodular T- / NK cell lymphoma, enteropathy type T cell lymphoma, hepatosplenic T cell lymphoma, astrocytic large cell lymphoma (ALCL), angiocetric And angioimmunoblastic T cell lymphoma, Mycosis fungoides / Sézary syndrome, cutaneous T cell lymphoma (CTCL)), Hodgkin Small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), lung adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma , Non glioma, acoustic schwanoma, craniopharyngioma, medulloblastoma, meningioma, pineoblastoma, pineoblastoma, primary brain lymphoma, germ cell tumor, superficial colon cancer, Tumor type colon cancer, ulcer infiltrating type colon cancer, diffuse infiltrating type colon cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, superficial expanded melanoma, nodular melanoma, malignant lentigo melanoma, terminal verruca Scleroderma tumor, carcinoid, pancreatic islet cell tumor, tumor type gastric cancer, ulcer limited local gastric cancer, ulcer invasive gastric cancer, gastric cancer, gastric cancer, gastric cancer, gastric cancer, Diffuse infiltrating gastric cancer, hepatocellular carcinoma and hepatoblastoma, epithelial ovarian carcinoma, borderline tumor , Germ cell tumor, interstitial tumor, serous adenocarcinoma, clear cell adenocarcinoma, endometrial adenocarcinoma, transitional cell carcinoma, mucinous adenocarcinoma, mixed ovarian cancer, squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma, But are not limited to, renal adenocarcinoma, adrenal gland, renal fibrosarcoma, transitional cell carcinoma (renal pelvis and / or ureter (uterer)), mastocytoma, perianal adenoma, perianal adenocarcinoma.
[0036]
　The subject (animal) to be treated is preferably a mammal, more preferably a mammal including an animal raised in a primate, a pet animal, a zoo or the like, a livestock animal, a competitive animal and the like , Particularly preferably human, dog or cat.
[0037]
　The route of administration of the immunity-inducing agent of the present invention to the living body may be oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration or the like is preferable. When the immunity-inducing agent is used for the purpose of cancer treatment, it can also be administered to the regional lymph nodes in the vicinity of the tumor to be treated, in order to enhance the anticancer effect. The dosage may be an amount effective for inducing immunity, so long as it is an amount effective for the treatment and / or prevention of cancer as long as it is used for treatment and / or prevention of cancer, for example. The effective amount for treating and / or preventing cancer is appropriately selected according to the size and symptoms of the tumor, but it is usually 0.0001 to 1000 μg as an effective amount per day for the target animal, It is in the range of 001 to 1000 μg, and it can be administered once or several times. Preferably, it is divided into several doses and administered every several days to several months. As specifically shown in the Examples below, the immunity inducing agent of the present invention can regress the tumor. Therefore, since it can exert anti-cancer action also on a small number of cancer cells in the early stage of development, it can be prevented before the onset of cancer or after cancer treatment, cancer onset and recurrence can be prevented. That is, the immunity-inducing agent of the present invention is useful for both treatment and prevention of cancer.
[0038]
　The immunity-inducing agent of the present invention may consist of only polypeptides, or may be appropriately mixed with pharmacologically acceptable additives such as a pharmacologically acceptable carrier, diluent, excipient and the like suitable for each administration mode It can also be formulated. The formulation method and usable additives are well known in the field of pharmaceutical preparations and any method and additives can be used. Specific examples of additives include diluents such as physiological buffer; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine and the like; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth and the like ; Lubricants such as magnesium stearate, polyethylene glycol, talc and silica, and the like, but are not limited thereto. Examples of formulations include oral preparations such as tablets, capsules, granules, powders and syrups, parenteral preparations such as inhalants, injections, suppositories and solutions. These preparations can be prepared by generally known manufacturing methods.
[0039]
　The immunity inducing agent of the present invention can be used in combination with an immunopotentiating agent capable of enhancing an immunological response in vivo. The immunopotentiating agent may be contained in the immunity inducing agent of the present invention or may be administered to the patient in combination with the immunity inducing agent of the present invention as a separate composition.
[0040]
　An example of the above-mentioned immunopotentiator is an adjuvant. Adjuvants can enhance the anticancer effect because they can enhance the immunological response by providing antigen reservoir (extracellular or macrophage), activating macrophages and stimulating specific sets of lymphocytes be able to. Therefore, in particular, when the immunity-inducing agent of the present invention is used for the treatment and / or prevention of cancer, it is preferable that the immunity-inducing agent further contains an adjuvant in addition to the polypeptide as the active ingredient. Numerous types of adjuvants are well known in the art and any adjuvant can be used.
[0041]
　Specific examples of adjuvants include, but are not limited to, MPL (SmithKline Beecham), the same obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide of Salmonella; QS21 (SmithKline Beecham), purified from Quillja saponaria extract QS-17, QS-17, QS-18 and QS-L1 (Soo, 10 others, "Moleculars and Cell (QS-17)" described in WO 96/33739 (SmithKline Beecham) Molecules and cells ", 1997, Vol. 7, p. 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; vitamin E; Montanide; Poly IC and its derivatives (poly ICLC etc), see also CpG oligonucleotides (eg Kreig, 7 others, "Nature", 374, 546-549); and Various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol. Among them, Freund's incomplete adjuvant, montanide, poly IC and derivatives thereof, CpG oligonucleotide are preferred. The mixing ratio of the adjuvant and the polypeptide is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably about 1: 1. However, the adjuvant is not limited to the above examples, and other adjuvants known in the art can also be used in administering the immunity-inducing agent of the present invention (see, for example, Goding, "Monoclonal antibodies: Principles and Practice ", Second Edition, 1986). Methods for preparing mixtures or emulsions of polypeptides and adjuvants are well known to those skilled in the art of vaccination.
[0042]
　In addition to the above adjuvants, factors that stimulate the subject's immune response can also be used as the immunopotentiating agent. For example, various cytokines having characteristics of stimulating lymphocytes or antigen presenting cells can be used in combination with the immunity inducing agent of the present invention as an immunopotentiating agent. Numerous cytokines capable of potentiating such immunological responses are known to those skilled in the art, examples of which include interleukin-12 (IL-12), which has been shown to enhance the protective effect of the vaccine, But are not limited to, GM-CSF, IL-18, interferon alpha, interferon beta, interferon omega, interferon gamma and Flt3 ligand. Such factors can also be used as the immunopotentiating agent and can be administered to a patient in combination with the immunity-inducing agent of the present invention either in the immunity-inducing agent of the present invention or as a separate composition.
[0043]
3. Antigen Presenting Cells or Cytotoxic T Cells
　The polypeptides can be presented to antigen presenting cells by contacting the polypeptides with antigen presenting cells (from a subject) ex vivo or in vitro. That is, the polypeptides (a) to (d) can be used as a treatment agent for antigen presenting cells. Here, as antigen presenting cells, dendritic cells or B cells carrying MHC class I molecules can be preferably used. A variety of MHC class I molecules have been identified and are well known. The MHC molecule in humans is called HLA. Examples of HLA class I molecules include HLA-A, HLA-B and HLA-C, more specifically HLA-A1, HLA-A0201, HLA-A0204, HLA- A0205, HLA- A0206, HLA-A 0207, HLA-A 11, HLA-A 24, HLA-A 31, HLA-A 6801, HLA-B 7, HLA-B 8, HLA-B 2705, HLA-B 37, HLA-Cw 0401, HLA-Cw 0602 and the like.
[0044]
　Dendritic cells or B cells carrying MHC class I molecules can be prepared from peripheral blood by well-known methods. For example, dendritic cells are induced from bone marrow, umbilical cord blood or patient peripheral blood using granulocyte macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4) , It is possible to induce tumor-specific dendritic cells.
[0045]
　Administering an effective amount of this dendritic cell can induce a desired response in the treatment of cancer. Bone marrow and umbilical cord blood provided by a healthy person, bone marrow or peripheral blood of the patient himself or herself can be used as cells to be used, but when using the original autologous cells of the patient, it is highly safe and has serious side effects It can also be expected to avoid it. Peripheral blood or bone marrow may be fresh, cryopreserved or cryopreserved. For peripheral blood, whole blood may be cultured, or only leukocyte components may be separated and cultured, but the latter is more efficient and preferable. Furthermore, among the leukocyte components, mononuclear cells may be separated. In addition, when originating from bone marrow or umbilical cord blood, the whole cells constituting the bone marrow may be cultured, or mononuclear cells may be separated and cultured therefrom. Peripheral blood and its white blood cell components and bone marrow cells include mononuclear cells, hematopoietic stem cells or immature dendritic cells and CD4 positive cells, which are the origin of dendritic cells. The cytokine used is not limited as long as it is a natural type or genetically modified type as long as it has characteristics confirmed safety and physiological activity, but preferably the quality to be used for medical use is ensured The standard sample is used in the minimum required amount. The concentration of the cytokine to be added is not particularly limited as long as it is a concentration at which dendritic cells are induced, and is usually about 10 to 1000 ng / mL in terms of the total concentration of cytokines, more preferably about 20 to 500 ng / mL. Culturing can be carried out using a well-known medium usually used for culturing leukocytes. The culturing temperature is not particularly limited as long as leukocyte proliferation is possible, but most preferably about 37 ° C., which is the human body temperature. In addition, the gas environment during cultivation is not particularly limited as long as leukocyte proliferation is possible, but it is preferably 5% CO 2Is preferably ventilated. Further, the culturing period is not particularly limited as long as the necessary number of cells are induced, but it is usually carried out between 3 days and 2 weeks. Appropriate suitable equipment can be used for the cell separation and culture, but it is preferable that safety is confirmed for medical use, the operation is stable and convenient. In particular, regardless of general containers such as petri dishes, flasks, bottles, etc., it is also possible to use a laminated container, a multistage container, a roller bottle, a spinner type bottle, a bag type incubator, a hollow fiber column, etc. .
[0046]
　The method of bringing the polypeptide and the antigen presenting cell into contact with each other ex vivo or in vitro can be carried out by well-known methods. For example, the antigen-presenting cells can be cultured in a culture medium containing the polypeptide. The peptide concentration in the medium is not particularly limited, but is usually about 1 to 100 μg / ml, preferably about 5 to 20 μg / ml. The cell density at the time of culturing is not particularly limited, but is usually about 10 3 to 10 7 cells / ml, preferably about 5 × 10 4 to 5 × 10 6 cells / ml. Culturing is preferably carried out in an atmosphere of 37 ° C. and 5% CO 2 according to a conventional method . The length of peptides that antigen presenting cells can present on the surface is usually about 30 amino acid residues at maximum. Accordingly, although not particularly limited, when the antigen-presenting cell and the polypeptide are brought into contact with each other ex vivo or in vitro, the polypeptide may be prepared to have a length of about 30 amino acid residues or less.
[0047]
　By culturing the antigen presenting cells in the presence of the polypeptide, the peptide is taken up by the MHC molecule of the antigen presenting cell and displayed on the surface of the antigen presenting cell. Therefore, using the above polypeptide, isolated antigen presenting cells containing a complex of the polypeptide and MHC molecule can be prepared.
[0048]
　Thus, the present invention also provides an antigen presentation comprising a conjugate of said polypeptide and an MHC molecule, comprising contacting said polypeptide with an antigen-presenting cell derived from a subject ex vivo or in vitro Providing a cell.
[0049]
　The present invention also provides an antigen presenting cell characterized by comprising a complex of the polypeptide and MHC molecule obtained by this method.
[0050]
　By contacting an antigen presenting cell comprising the polypeptide and a complex of the MHC molecule prepared as described above with the T cell ex vivo or in vitro, the cytotoxic T Cells can be induced and expanded. This can be carried out by co-culturing the antigen-presenting cells and the T cells in a liquid medium. For example, the antigen presenting cells can be suspended in a liquid medium, placed in a container such as a well of a microplate, and T cells are added thereto for culturing. Although the mixing ratio of antigen presenting cells and T cells during co-cultivation is not particularly limited, it is usually about 1: 1 to 1: 100, preferably about 1: 5 to 1: 20 in the ratio of the number of cells. The density of the antigen presenting cells suspended in the liquid medium is not particularly limited, but is usually about 100 to 10,000,000 cells / ml, preferably about 10,000 to 1 million cells / ml. Co-cultivation is preferably carried out in an atmosphere of 37 ° C. and 5% CO 2 according to a conventional method . The cultivation time is not particularly limited, but it is usually about 2 days to 3 weeks, preferably about 4 days to 2 weeks. In addition, co-culturing is preferably carried out in the presence of one or more interleukins such as IL-2, IL-6, IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5 to 20 U / ml, the concentration of IL-6 is usually about 500 to 2000 U / ml, the concentration of IL-12 is usually about 5 to 20 ng / ml However, it is not limited to them. The co-cultivation described above may be repeated once or several times with the addition of fresh antigen-presenting cells. For example, the operation of discarding the culture supernatant after co-cultivation, adding a suspension of fresh antigen-presenting cells and further performing co-cultivation may be repeated once or several times. Conditions for each co-cultivation may be the same as above.
[0051]
　By the co-cultivation described above, cytotoxic T cells specific for the polypeptide are induced and proliferated. Thus, the polypeptide can be used to prepare isolated T cells that selectively bind to the complex of the polypeptide and the MHC molecule.
[0052]
　Thus, the present invention further provides a method of treating a cellular disorder specific to a polypeptide as described above, comprising contacting the antigen-presenting cell with a subject-derived T cell ex vivo or in vitro to activate the T cell Thereby providing a method for producing sex T cells.
[0053]
　The present invention also provides cytotoxic T cells obtained by this method and specific for the polypeptide described above.
[0054]
　As described in the Examples described later, the MRAP2 gene is expressed in the leukemia, malignant lymphoma, lung cancer, brain tumor, colon cancer, melanoma, neuroblastoma, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma Or perianal adenocarcinoma, as shown in Fig. Therefore, it is considered that in these cancer types, MRAP2 is present significantly more than normal cells. When cytotoxic T cells prepared as described above are administered in vivo so that a part of the polypeptide of MRAP2 present in cancer cells is presented to MHC molecules on the surface of cancer cells, Cytotoxic T cells can damage cancer cells as markers. In addition, antigen-presenting cells presenting a part of the polypeptide of MRAP2 can induce and proliferate cytotoxic T cells specific for the polypeptide also in vivo, so that the antigen presenting cells can be produced By administering it into the body, cancer cells can also be damaged. That is, the cytotoxic T cells and the antigen presenting cells prepared using the polypeptide are also useful as agents for the treatment and / or prophylaxis of cancer, similarly to the immunity inducing agent of the present invention.
[0055]
　In the case of administering the above isolated antigen-presenting cells or isolated T cells to a subject, in order to avoid an immune response in vivo which attacks these cells as a foreign body, the antigen collected from the patient to be treated It is preferable that the presenting cells or T cells are prepared using the polypeptides (a) to (d) as described above.
[0056]
　The route of administration of a therapeutic and / or prophylactic agent for cancer comprising isolated antigen-presenting cells or isolated T cells as an active ingredient is preferably parenteral administration such as intravenous administration or intraarterial administration. The dosage is appropriately selected according to the symptoms, the purpose of administration and the like, but it is generally 1 to 10 trillion, preferably 1 million to 1 billion, and this is administered once every several days to several months It is preferable to administer it. The formulation may be, for example, a suspension of the cells in physiological buffered saline, etc., and can be used in combination with other anticancer agents, cytokines, and the like. One or more additives well known in the pharmaceutical field can also be added.
[0057]
4. DNA Vaccine By
　expressing the polynucleotide encoding the polypeptide of (a) to (d) in the body of the target animal, antibody production and cytotoxic T cell can be induced in the living body , An effect equivalent to administration of the polypeptide can be obtained. That is, the immunity-inducing agent of the present invention comprises a polynucleotide encoding the polypeptides (a) to (d) above and contains a recombinant vector capable of expressing the polypeptide in vivo as an active ingredient It may be. As shown in Examples described later, a recombinant vector capable of expressing such an antigen polypeptide is also called a DNA vaccine.
[0058]
　The vector used for producing the DNA vaccine is not particularly limited as long as it is a vector that can be expressed in a target animal cell (preferably a mammalian cell), and may be a plasmid vector or a viral vector, which is publicly known in the field of DNA vaccine Any vector may be used. As described above, polynucleotides such as DNA and RNA encoding the polypeptide can be easily prepared by a conventional method. Incorporation of the polynucleotide into the vector can also be carried out using methods well known to those skilled in the art.
[0059]
　The administration route of the DNA vaccine is preferably a parenteral administration route such as intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration and the like, and the dosage can be appropriately selected according to the type of antigen and the like , Usually about 0.1 μg to 100 mg, preferably about 1 μg to 10 mg, as the weight of the DNA vaccine per kg of body weight.
[0060]
　As a method using viral vectors, for example, RNA viruses such as retrovirus, adenovirus, adeno-related (also referred to as "adeno-associated") virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus or DNA virus or DNA virus , A method of incorporating a polynucleotide encoding the above polypeptide and infecting the target animal. Among them, a method using retrovirus, adenovirus, adeno-associated virus, vaccinia virus or the like is particularly preferable.
[0061]
　Other methods include direct administration of the expression plasmid intramuscularly (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method, etc. In particular, DNA vaccine method, liposome Method is preferred.
[0062]
　In order to allow the gene encoding the above polypeptide used in the present invention to act as a medicine in practice, in vivo methods of directly introducing the gene into the body, a method of collecting a certain kind of cells from the target animal, (Nikkei Science, April 1994, p. 20 - 45, Monthly Pharmaceutical Affairs, 1994, Vol. 36, No.1, pp. 23-48, Experimental Medicine Extra Publication, 1994, Volume 12, No. 15, and cited references etc.), an in vivo method is more preferable.
[0063]
　When administered by an in vivo method, it can be administered by an appropriate administration route according to diseases, symptoms and the like for therapeutic purposes. For example, it can be administered intravenously, arterially, subcutaneously, intramuscularly, or the like. In the case of administration according to an in vivo method, for example, it may take the form of a solution or the like, but it is generally considered to be an injection containing DNA encoding the polypeptide of the present invention which is an active ingredient, , A conventional carrier may be added. In the case of liposomes or membrane-fused liposomes (Sendai virus (HVJ) -liposome etc.) containing the DNA, it can be in the form of a liposome preparation such as a suspension agent, a cryoprotectant, a centrifugal concentration frozen agent and the like.
[0064]
　In the present invention, for example, the phrase "the nucleotide sequence represented by SEQ ID NO: 1" includes the nucleotide sequence actually shown in SEQ ID NO: 1, as well as a sequence complementary thereto. Therefore, for example, when referring to "a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1", a single stranded polynucleotide having the nucleotide sequence actually shown in SEQ ID NO: 1, its complementary nucleotide sequence , And double-stranded polynucleotides comprising them. When a polynucleotide encoding the polypeptide used in the present invention is prepared, any nucleotide sequence is appropriately selected, but those skilled in the art can easily select the nucleotide sequence.
Example
[0065]
　Hereinafter, the present invention will be described more specifically based on examples.
[0066]
Example 1 Acquisition of Novel Cancer Antigen Protein by SEREX Method
(1) Preparation of cDNA Library
　Total RNA was extracted from canine testis by acid-guanidium-phenol-chloroform method (Acid guanidium-Phenol-Chloroform method) Poly A RNA was purified using the Oligotex-dT30 mRNA purification kit (Takara Shuzo Co., Ltd.) according to the protocol attached to the kit.
[0067]
　A cDNA phage library was synthesized using this obtained mRNA (5 μg). For preparation of cDNA phage libraries, libraries were prepared using cDNA Synthesis kit, Zap-cDNA Synthesis Kit, ZAP-cDNA Gigapack III Gold Cloning Kit (STRATAGENE) according to the protocol attached to the kit. The size of the prepared cDNA phage library was 1 × 10 6 pfu / ml.
[0068]
(2) Screening of cDNA library with serum
　Immunoscreening was carried out using the cDNA phage library prepared above. Specifically, it infects Escherichia coli host (XL1-Blue MRF ') to an NZY agarose plate of Φ 90 × 15 mm to become approximately 2500 clones and cultures at 42 ° C. for 3 to 4 hours to make lysed plaques , The plate was covered with a nitrocellulose membrane (Hybond C Extra: manufactured by GE Healthecare Bio-Sciences) permeated with IPTG (isopropyl-β-D-thiogalactoside) at 37 ° C. for 4 hours to induce and express the protein, Protein was transferred to the membrane. Thereafter, the membrane was recovered and immersed in TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% skim milk powder and shaken overnight at 4 ° C. to suppress non-specific reaction. This filter was reacted with Canine Serum diluted 500-fold at room temperature for 2 to 3 hours.
[0069]
　As the above-mentioned canine serum, serum collected from a dog of leukemia was used. These sera were stored at -80 ° C. and pretreated immediately before use. The serum pretreatment method is as follows. That is, host Escherichia coli (XL1-BLue MRF ') was infected with λ ZAP Express phage without the insertion of a foreign gene and then cultured overnight at 37 ° C. on an NZY plate medium. Next, a buffer of 0.2 M NaHCO 3　 pH 8.3 containing 0.5 M NaCl was added to the plate and the plate was allowed to stand at 4 ° C. for 15 hours, and then the supernatant was collected as an E. coli / phage extract. Next, the recovered E. coli / phage extract was passed through NHS-column (manufactured by GE Healthecare Bio-Science) to immobilize proteins derived from Escherichia coli and phage. Dogs serum was passed through the protein immobilized column and reacted, and antibodies adsorbing to E. coli and phage were removed from the serum. The serum fraction passing through the column was diluted 500-fold with TBS containing 0.5% skimmed milk powder and used as an immuno-screening material.
[0070]
　The membranes blotted with such treated sera and proteins were washed 4 times with TBS-T (0.05% Tween 20 / TBS), then washed 5 times with TBS containing 0.5% skimmed milk powder as a secondary antibody Goat anti-dog IgG (Goat anti Dog IgG-h + L HRP conjugated: manufactured by BETHYL Laboratories) diluted was reacted at room temperature for 1 hour and detected by enzyme color reaction using NBT / BCIP reaction solution (manufactured by Roche) Colonies corresponding to the chromogenic reaction positive site were collected from a Φ 90 × 15 mm NZY agarose plate 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) . The secondary and tertiary screening are repeated in the same manner as above until the chromogenic positive colonies are unified, and approximately 10,000 phage clones reacting with IgG in the serum are screened, and one positive clone is treated as a single I got away.
[0071]
(3) Sequence Identity Search of Isolated Antigen Gene In order to subject
　one positive clone isolated by the above method to a nucleotide sequence analysis, an operation of converting from a phage vector into a plasmid vector was performed. Specifically, 200 μl of a solution prepared from host Escherichia coli (XL 1-Blue MRF ') to have an absorbance OD 600 of 1.0, 100 μl of the purified phage solution and 1 μl of ExAssist helper phage (manufactured by STRATAGENE) were mixed, ° C for 15 minutes, add 3 ml of LB medium, incubate at 37 ° C for 2.5 to 3 hours, immediately incubate in a water bath at 70 ° C for 20 minutes, then centrifuge at 4 ° C and 1000 x g for 15 minutes , And the supernatant was recovered as a phagemid solution. Next, 200 μl of a phagemid host Escherichia coli (SOLR) adjusted to have an absorbance OD 600 of 1.0 and 10 μl of the purified phage solution were mixed and reacted at 37 ° C. for 15 minutes, 50 μl of which was added to ampicillin (final concentration of 50 μg / ml ) Containing LB agar medium and cultured at 37 ° C. overnight. A single colony of transformed SOLR was collected and cultured at 37 ° C. in an LB medium containing ampicillin (final concentration 50 μg / ml), and then plasmid DNA having the desired insert was purified using QIAGEN plasmid Miniprep Kit (Qiagen) did.
[0072]
　The purified full-length sequence of the insert was analyzed by the primer walking method using the T3 primer set forth in SEQ ID NO: 9 and the T7 primer set forth in SEQ ID NO: 10. By this sequence analysis, the gene sequence shown in SEQ ID NO: 3 was obtained. Using the nucleotide sequence and amino acid sequence of this gene, sequence identity search program BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) was performed to search sequence identity with known genes As a result, the obtained gene was found to be MRAP2 gene. In human MRAP2 which is a human homologous factor of canine MRAP2, the sequence identity is 91% of the base sequence and 94% of the amino acid sequence, and in the feline homologous factor feline MRAP 2 the sequence identity is 95% of the base sequence and 96% of the amino acid sequence There was 84% ​​of the nucleotide sequence and 88% of the amino acid sequence in the murine homologous factor mouse MRAP2. The nucleotide sequence of human MRAP2 is shown in SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2, the nucleotide sequence of feline MRAP2 is SEQ ID NO: 5, the amino acid sequence is SEQ ID NO: 6, the nucleotide sequence of mouse MRAP2 is SEQ ID NO: 7, the amino acid sequence is sequence It is shown in number 8.
[0073]
(4) Analysis of Gene Expression in Each Tissue Expression in
　the various normal tissues, various tumor tissues, and various cancer cell lines of dogs, humans and mice is subjected to RT-PCR (Reverse Transcription-PCR) method . The reverse transcription reaction was carried out as follows. That is, 50 to 100 mg of each tissue and 5 to 10 × 10 6Total RNA was extracted from the cells using TRIZOL reagent (manufactured by Life technology) according to the attached protocol. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Life technology) according to the attached protocol. Gene pool cDNA (manufactured by Life technology), QUICK-Clone cDNA (Clontech) and Large-Insert cDNA Library (manufactured by Clontech) were used as cDNAs of human normal tissues (brain, testis, colon, placenta). The PCR reaction was carried out as follows using the obtained gene-specific primers (the canine primers are SEQ ID NOs: 11 and 12, the human primers are SEQ ID NOs: 13 and 14, and the mouse primer is SEQ ID NOs: 15 and 16) as follows. That is, 0.25 μl of the sample prepared by the reverse transcription reaction, 2 μM of each of the above primers, 0.2 mM of each dNTP, 0.65 U of ExTaq polymerase (Takara Shuzo Co., Ltd.) was added to each reagent and attached buffer, And the cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 30 times using a Thermal Cycler (manufactured by BIO RAD). As a result, as shown in FIG. 1, strong expression of the canine MRAP2 gene was observed in canine tumor tissues in mastocytoma and perianal adenocarcinoma (FIG. 1). In human MRAP2 gene expression, expression is not found in most tissues in healthy human tissues, whereas in human cancer cells it is not expressed in leukemia, malignant lymphoma, lung cancer, brain tumor, colon cancer, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophagus Strong expression was found in cell lines of cancer and kidney cancer (FIG. 2). In addition, the expression of the mouse MRAP2 gene was detected in leukemia, melanoma, neuroblastoma cell lines (FIG. 3).
[0074]
(Example 2) Analysis of cancer antigenicity in vivo of MRAP2
(1) Preparation of recombinant vector expressing mouse MRAP2 in vivo Based on
　the base sequence of SEQ ID NO: 7, in vivo To prepare a recombinant vector expressing mouse MRAP2. PCR was prepared from mouse leukemia cell line EL4 (purchased from ATCC) in which expression was observed in Example 1. 1 μl each of cDNA, 0.4 μM of each of two kinds of primers (described in SEQ ID NOs: 17 and 18) containing EcoRI and NotI restriction enzyme cleavage sequences, 0.2 μM each of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) containing 0.2 mM dNTP, Each reagent and attached buffer were added to make a total volume of 50 μl and a cycle of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C. for 1 minute was repeated 30 times using a Thermal Cycler (manufactured by BIO RAD) I did it by repeating. In addition, the above two types of primers amplified the region encoding the full length amino acid sequence of SEQ ID NO: 8. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel and a DNA fragment of about 600 bp was purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN).
[0075]
　The purified DNA fragment was ligated to the cloning vector pCR-Blunt (Life technology). After transformation into E. coli, the plasmid was recovered, and it was confirmed by the sequence that the amplified gene fragment coincided with the target sequence. The plasmid consistent with the sequence of interest was treated with EcoRI and NotI restriction enzymes, purified with QIAquick Gel Extraction Kit, and the desired gene sequence was inserted into the mammalian expression vector pcDNA 3.1 (Invitrogen) treated with EcoRI and NotI restriction enzymes (Hereinafter referred to as mouse MRAP2 / pcDNA 3.1). By using this vector mouse MRAP2 protein is produced in mammalian cells.
[0076]
　50 μg of gold particles (manufactured by Bio Rad), 100 μl of spermidine (manufactured by SIGMA) and 100 μl of 1 M CaCl 2　 (manufactured by SIGMA) were added to 100 μg of the plasmid DNA prepared above, and the mixture was stirred by vortexing and allowed to stand at room temperature for 10 minutes (Hereinafter referred to as gold-DNA particles). After centrifugation at 3000 rpm for 1 minute, the supernatant was discarded and washed 3 times with 100% ethanol (Wako). 6 ml of 100% ethanol was added to the gold-DNA particles and sufficiently stirred by vortexing, gold DNA particles were poured into Tefzel Tubing (manufactured by Bio Rad) and precipitated on the wall. Tefzel Tubing ethanol with gold-DNA particles attached was air dried and cut to length suitable for gene gun (hereinafter referred to as mouse MRAP 2 / tube).
[0077]
(2) Antitumor effect of mouse MRAP 2 by DNA vaccination method 1
　tube (mouse MRAP 2 / tube) prepared above was injected to 5 A / J mice (7 weeks old, male, purchased from Japan SLC) Was immobilized on a gene gun and DNA vaccine was transdermally administered to the peritoneal cavity of a shaved mouse at a pressure of 400 psi for a total of three times every 7 days using pure helium gas (the amount of inoculation of plasmid DNA was 2 μg / mouse ) The murine leukemia cell line EL4 cells showing the expression of the MRAP2 gene in Example 1 were transplanted and the antitumor effect was evaluated (preventive model). As a control, five mice were administered with a preventive model of plasmid DNA in which the mouse MRAP2 gene had not been inserted.
[0078]
　Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. As a result of this study, in the preventive model, marked regression of the tumor was observed in the mouse MRAP2 plasmid-administered group in the control group and the mouse MRAP2 plasmid administration group after 28 days, respectively, to 1359 mm 3 , 601 mm 3 . In addition, as a result of observing the course of survival, in the preventive model, all cases died on the 61st day after administration in the control group, while 60% of the mice survived in the mouse MRAP2 plasmid administered group. From the above results, a significant antitumor effect was observed in the mouse MRAP2 plasmid-administered group as compared with the control group.
[0079]
(3) Antitumor effect of mouse MRAP 2 by DNA vaccine method - The
　tube (mouse MRAP 2 / tube) prepared above was administered to 5 A / J mice (7 weeks old, male, purchased from Japan SLC) Was immobilized on a gene gun and DNA vaccine was transdermally administered to the peritoneal cavity of a shaved mouse at a pressure of 400 psi for a total of three times every 7 days using pure helium gas (the amount of inoculation of plasmid DNA was 2 μg / mouse ) The mouse melanoma cell line B16 cell showing expression was transplanted in Example 1 to evaluate the antitumor effect (preventive model). As a control, five mice were administered with a preventive model of plasmid DNA in which the mouse MRAP2 gene had not been inserted.
　Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. The results of this study, the prevention model, after 28 days, the control group and mice MRAP2 plasmid administration group, respectively, 936Mm 3 , 483 mm 3 , and the noticeable regression of tumors in mice MRAP2 plasmid treated group was observed. In addition, as a result of observing the course of survival, in the preventive model, all cases died on the 68th day after administration in the control group, whereas 60% of the mice survived in the mouse MRAP2 plasmid administered group. From the above results, a significant antitumor effect was observed in the mouse MRAP2 plasmid-administered group as compared with the control group.
[0080]
(4) Preparation of recombinant vector expressing human MRAP2 in vivo Based on
　the nucleotide sequence of SEQ ID NO: 1, a recombinant vector expressing human MRAP2 in vivo was prepared in vivo by the following method. PCR was prepared from the human leukemia cell line K562 (purchased from ATCC) in which expression was observed in Example 1. 1 μl of cDNA, 0.4 μM of each of two kinds of primers (described in SEQ ID NOs: 19 and 20) containing EcoRI and NotI restriction enzyme cleavage sequences, 0.2 μM each of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) with 0.2 mM dNTP, 1.25 U Each reagent and attached buffer are added so that the total amount is 50 μl and the cycle of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C. for 1 minute is repeated 30 times using Thermal Cycler (manufactured by BIO RAD) By doing that. The above two types of primers amplified the region encoding the full length amino acid sequence of SEQ ID NO: 2. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel and a DNA fragment of about 600 bp was purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN).
[0081]
　The purified DNA fragment was ligated to the cloning vector pCR-Blunt (Life technology). After transformation into E. coli, the plasmid was recovered, and it was confirmed by the sequence that the amplified gene fragment coincided with the target sequence. The plasmid consistent with the sequence of interest was treated with EcoRI and NotI restriction enzymes, purified with QIAquick Gel Extraction Kit, and the desired gene sequence was inserted into the mammalian expression vector pcDNA 3.1 (Invitrogen) treated with EcoRI and NotI restriction enzymes (Hereinafter referred to as human MRAP2 / pcDNA 3.1). By using this vector human MRAP2 protein is produced in mammalian cells.
[0082]
　50 μg of gold particles (manufactured by Bio Rad), 100 μl of spermidine (manufactured by SIGMA) and 100 μl of 1 M CaCl 2　 (manufactured by SIGMA) were added to 100 μg of the plasmid DNA prepared above, and the mixture was stirred by vortexing and allowed to stand at room temperature for 10 minutes (Hereinafter referred to as gold-DNA particles). After centrifugation at 3000 rpm for 1 minute, the supernatant was discarded and washed 3 times with 100% ethanol (Wako). 6 ml of 100% ethanol was added to the gold-DNA particles and sufficiently stirred by vortexing, gold DNA particles were poured into Tefzel Tubing (manufactured by Bio Rad) and precipitated on the wall. Tefzel Tubing ethanol with gold-DNA particles attached was air dried and cut to length suitable for gene gun (hereinafter referred to as human MRAP 2 / tube).
[0083]
(5) Establishment of full-length human MRAP2 steady-expressing cells
　Human MRAP2 / pcDNA 3.1 prepared as described above was introduced into mouse neuroblastoma cell line N2a cell (ATCC) by lipofection and 500 μg / ml of G418 (Nacalai ), An N2a cell line that constantly expresses full-length human MRAP2 was established (N2a-human MRAP2). In addition, an expression vector into which cDNA encoding human MRAP2 was not inserted (hereinafter referred to as emp / pcDNA 3.1) was introduced in the same manner as above to select cells selected as control cells (hereinafter referred to as N2a-emp).
[0084]
(6) Antitumor effect of human MRAP2 by DNA vaccine method The
　tube (human MRAP2 / tube) prepared above was transfected with 5 A / J mice (7 weeks old, male, purchased from Japan SLC) using the gene DNA vaccine was transdermally administered to the peritoneal cavity of a shaved mouse at a pressure of 400 psi for a total of three times every 7 days, fixed to a gun, and the abdominal cavity of a shaved mouse was transdermally administered (the amount of plasmid DNA inoculation reached 2 μg / mouse ) The anti-tumor effect was evaluated by transplanting N2a-human MRAP2 or N2a-emp prepared above (preventive model). As a control, 5 mice were administered with a preventive model of plasmid DNA into which the human MRAP2 gene had not been inserted.
[0085]
　Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. As a result of this study, in the prevention model of N2a-human MRAP2, after 28 days, the control group and the human MRAP2 plasmid administration group were 1379 mm 3 , 513 mm 3 respectively, and in the human MRAP 2 plasmid administration group, remarkable regression of the tumor was observed Was observed. In addition, as a result of monitoring the course of survival, 60% of the mice survived in the human MRAP2 plasmid-administered group, whereas in the control group all patients died at 61 days after administration. From the above results, in the prevention model of N2a-human MRAP2, a significant anti-tumor effect was observed in the human MRAP2 plasmid administered group as compared with the control group. On the other hand, in the prevention model of N2a-emp, no significant antitumor effect was observed in the human MRAP2 plasmid administered group as compared with the control group.
Industrial applicability
[0086]
　INDUSTRIAL APPLICABILITY The present invention provides an immunity inducing agent comprising a polypeptide exhibiting antitumor activity against various cancers, and is therefore useful for treatment and / or prevention of cancer.
　All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.
The scope of the claims
[Claim 1]
　At least one polypeptide selected from the following polypeptides (a) to (d) and having immunity-inducing activity, or a polynucleotide encoding the polypeptide, and capable of expressing the polypeptide in vivo A recombinant vector, as an active ingredient.
(A) a polypeptide consisting of at least 7 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing up to the full length of the polypeptide
(b) SEQ ID NO: 2, 4, 6 , Or a polypeptide consisting of an amino acid sequence in which 1 to several amino acids are deleted, substituted or added in the amino acid
sequence represented by SEQ ID NO: 2, 4, 6, or 8 in the sequence listing (c) A polypeptide comprising an amino acid sequence having 90% or more sequence identity to the sequence
(d) a polypeptide comprising as a partial sequence any of the polypeptides (a) to (c)
[Claim 2]
　2. The immunity inducing agent according to claim 1, which is a treating agent for antigen presenting cells.
[Claim 3]
　The immunity-inducing agent according to claim 1, which is an active ingredient of a therapeutic and / or prophylactic agent for cancer.
[Claim 4]
　4. The immunity inducing agent according to claim 3, wherein the cancer is a cancer expressing MRAP2.
[Claim 5]
　Claim 1, wherein the cancer is leukemia, malignant lymphoma, lung cancer, brain tumor, colon cancer, melanoma, neuroblastoma, pancreatic cancer, gastric cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma or perianal adenocarcinoma The immunity inducing agent according to 3 or 4.
[Claim 6]
　The immunity inducing agent according to any one of claims 1 to 5, further comprising an immunopotentiating agent.
[7]
　Wherein the immunopotentiator is selected from the group consisting of Freund's incomplete adjuvant, montanide, poly IC and its derivatives, CpG oligonucleotide, interleukin 12, interleukin 18, interferon alpha, interferon beta, interferon omega, interferon gamma, and Flt3 ligand 7. The immunity-inducing agent according to claim 6, which is at least one selected from:
[Claim 8]
　A method for producing an antigen-presenting cell comprising a complex of said polypeptide and MHC molecule comprising contacting the polypeptide as defined in claim 1 with an antigen-presenting cell derived from a subject ex vivo or in vitro.
[Claim 9]
　9. The method of claim 8, wherein the antigen presenting cell is a dendritic cell or a B cell carrying an MHC class I molecule.
[Claim 10]
　10. The method of claim 1, comprising contacting antigen-presenting cells obtained by the method according to claim 8 or 9 with T cells derived from a subject ex vivo or in vitro to activate said T cells. Method for producing cytotoxic T cells specific for peptides.
[Claim 11]
　A pharmaceutical composition comprising at least one polypeptide selected from the following polypeptides (a) to (d) and having immunity inducing activity, or a polynucleotide encoding the polypeptide, which is capable of expressing the polypeptide in vivo A method for inducing immunity, comprising administering to a subject a recombinant vector.
(A) a polypeptide consisting of at least 7 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing up to the full length of the polypeptide
(b) SEQ ID NO: 2, 4, 6 , Or 8, a polypeptide consisting of an amino acid sequence in which 1 to several amino acids are deleted, substituted or added
(c) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8 A polypeptide comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence
(d) a polypeptide comprising as a partial sequence any of the polypeptides (a) to (c)