Entitled treatment and / or prevention a pharmaceutical composition for cancer

Technical field

[0001]

　The present invention relates to a novel pharmaceutical use as such treatment and / or prophylactic agent for cancer antibodies or fragments thereof against MRAP2.

Background technique

[0002]

　Recently, an antigen protein on the cancer cells were targeted, various antibody drugs for the treatment of cancer has emerged in the world. Although these therapeutic antibodies has been noted constant efficacy is obtained as a cancer-specific therapeutic agents, many of antigen protein to be targeted are those also expressed in a plurality of normal cells, the result of antibody administration, the cancer cells not only normal cells antigen is expressed will be impaired, side effects resulting is a problem. Thus, to identify cancer antigen specifically expressed in cancer cell surface, if it is possible to use a targeted antibody it as a pharmaceutical, it is expected to allow treatment with fewer side effects therapeutic antibodies.

[0003]

　Melanocortin 2 receptor accessory protein 2 (MRAP2) is type 1 or type 2 transmembrane protein, are involved in the activation control of the melanocortin receptor (MCR), which functions in energy metabolism in vivo (Non-Patent Document 1 ). Further, MRAP2 has been reported to become obese despite no overeating deficient mice, in severely obese patients in humans has been reported that some patients with mutations in MRAP2 gene (non-Patent Document 2). However, having an immunity-inducing activity MRAP2 protein on cancer cells and thereby no reports that the protein is useful in the treatment or prevention of cancer.

CITATION

Non-Patent Document

[0004]

Non-Patent Document 1: 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

Problems that the Invention is to Solve

[0005]

　An object of the present invention is to identify cancer antigen proteins specifically expressed on the surface of cancer cells, antibodies it targeted is to provide a use as therapeutic and / or prophylactic agent for cancer.

Means for Solving the Problems

[0006]

　The present invention intensively studied, by SEREX method using testis tissue-derived cDNA library and leukemia canine patient serum dog, encodes a protein that binds to antibodies present in serum from cancer-bearing living body get the cDNA, the obtained canine gene and its human, cat, based on mouse homology gene, to produce antibodies against MRAP2 and their MRAP2 having the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 . And MRAP2 leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, specifically expressed in mast cell tumors or perianal adenocarcinoma and that it, and some of MRAP2 protein was found to be expressed specifically in the cell surface thereof a cancer cell. Then, an antibody against the portion expressed on the cell surface of each cancer cell thereof MRAP2 is found that damaging cancer cells expressing MRAP2, and completed the present invention.

[0007]

　Accordingly, the present invention includes the following (1) to (11).
(1) SEQ ID NO: amino acid sequence represented by 2,4,6 or 8, or an amino acid sequence having the amino acid sequence 80% or more sequence identity, the MRAP2 protein, or 7 or more contiguous amino acids having a fragments, and comprising an antibody or fragment thereof having an immunological reactivity as an active ingredient, a pharmaceutical composition for the treatment and / or prevention of cancer, including.
(2) the MRAP2 protein a partial polypeptide of 7 or more contiguous amino acids of an amino acid sequence represented by any of the even-numbered SEQ ID NO: of SEQ ID NO: 10 to 24 polypeptide, or the amino acid comprising a polypeptide consisting of an amino acid sequence having a sequence at least 80% sequence identity, and the antibody or fragment thereof having an immunological reactivity as an active ingredient, a pharmaceutical composition according to (1).
(3) wherein the cancer is a cancer expressing MRAP2 on the cell surface The pharmaceutical composition according to (1) or (2).
(4) wherein the cancer is leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, the group consisting of mast cell tumors and perianal adenocarcinoma It is selected from (1) to the pharmaceutical composition according to any one of (3).
(5) wherein the antibody is a monoclonal antibody or a polyclonal antibody, (1) to the pharmaceutical composition according to any one of (4).
(6) wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody or multispecific antibody, (1) a pharmaceutical composition according to any one of - (5).
(7) The N-terminal partial polypeptide of MRAP2 protein, amino acid sequence represented by SEQ ID NO: 10, 14, and 18 or 22, or an amino acid sequence having the amino acid sequence 80% or more sequence identity, consisting antibodies or fragments thereof having a polypeptide immunologically reactive.
(8) a C-terminal partial polypeptide of MRAP2 protein, amino acid sequence represented by SEQ ID NO: 12, 16, 20 or 24, or an amino acid sequence having the amino acid sequence 80% or more sequence identity, consisting antibodies or fragments thereof having a polypeptide immunologically reactive.
(9) wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody or multispecific antibody, antibody or fragment thereof according to (7) or (8).
(10) (1) to the pharmaceutical composition according to any one of (6), comprising a pharmaceutical composition comprising an anti-tumor agent, a combination medicament for the treatment and / or prevention of cancer.
(11) SEQ ID NO: amino acid sequence represented by 2,4,6 or 8, or an amino acid sequence having the amino acid sequence 80% or more sequence identity, the MRAP2 protein, or 7 or more contiguous amino acids having a fragments thereof, and antibodies or fragments thereof having immunological reactivity comprising administering to a subject, the treatment and / or prevention of cancer including.

[0008]

　This description includes the disclosure of the priority document of the present application Japanese Patent Application No. 2016-064033.

Effect of the invention

[0009]

　Antibodies against MRAP2 used in the present invention, the failure of cancer cells. Thus, antibodies to MRAP2 are useful for the treatment or prevention of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

[Figure 1] dog MRAP2 gene was identified, a diagram showing the expression patterns in canine tumor tissue.
[Figure 2] of the identified MRAP2 gene, shows the expression pattern in various tissues and human cancer cell lines. A: Human MRAP2 gene, indicating the expression pattern of each human tissue. B: Human MRAP2 gene, indicating the expression pattern of each human cancer cell lines.
[3] identified murine MRAP2 gene, shows the expression pattern of each cancer cell line in mice.
[4] According to polyclonal antibody (anti-N-terminal side MRAP2 polyclonal antibody and an anti-C-terminal MRAP2 polyclonal antibody) against MRAP2, cytotoxic activity against leukemia cell lines expressing MRAP2 gene (K562) and malignant lymphoma cell lines (Namalwa) is a diagram illustrating a. Control -1; Control cytotoxic activity against K562 cells upon addition of a polyclonal antibody, anti-N-terminal side -1; cytotoxic activity against K562 cells upon addition of anti-N-terminal side MRAP2 polyclonal antibody, anti-C-terminal - 1; shows a cytotoxic activity against K562 cells upon addition of anti-C-terminal MRAP2 polyclonal antibodies. Controls-2; Control cytotoxic activity against Namalwa cells upon addition of a polyclonal antibody, anti-N-terminal side -2; cytotoxic activity against Namalwa cells upon addition of anti-N-terminal side MRAP2 polyclonal antibody, anti-C-terminal - 2; exhibit cytotoxic activity against Namalwa cells upon addition of anti-C-terminal MRAP2 polyclonal antibodies.

DESCRIPTION OF THE INVENTION

[0011]

　The present invention is an antibody or fragment thereof to MRAP2 protein or fragment thereof, preferably the antigen-binding fragments, for cancer treatment and / or prophylactic use.

[0012]

　The present invention comprises an amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, or the amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 99% or more, for example MRAP2 protein having an amino acid sequence, having the sequence identity 99.5%), or the overall length of not less than 7 consecutive (7 to each sequence, preferably 7 to 150, more fragment thereof preferably containing 7 to amino acids of 50), and comprising an antibody or fragment thereof having an immunological reactivity as an active ingredient, a pharmaceutical composition for the treatment and / or prevention of cancer.

[0013]

　The present invention also provides, MRAP2 protein a partial polypeptide, the total length of not less than 7 consecutive (7 to the sequence of the amino acid sequence represented by any one of the even-numbered SEQ ID NO: of SEQ ID NO: 10 to 24, preferably 7 to 40, more preferably from 7 to 20, for example, 7 to 12 or 8 polypeptide consisting of amino acids of the ~ 11), or the amino acid sequence having 80% or more (preferably at least 85% , more preferably 90% or more, more preferably 95% or more, particularly preferably an antibody or an active ingredient a fragment thereof having a sequence identity to a polypeptide consisting of an amino acid sequence having a immunological reactivity than 97%) including as relates to pharmaceutical compositions for the treatment and / or prevention of cancer.

[0014]

　Anti-tumor activity of the antibody or fragment thereof to the polypeptide or a fragment thereof comprising an amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 for use in the present invention, in vivo inhibition of tumor growth to a tumor-bearing animals ( by examining in in vivo), or, as will be described later, with respect to tumor cells expressing the polypeptide, whether the ex vivo indicate cytotoxic activity via immune cells or complement (in vitro) it can be evaluated by examining in. Similarly, antitumor activity of the antibody or fragment thereof to the polypeptide or fragment thereof of even SEQ ID NO: of SEQ ID NO: 10-24 for use in the present invention, in vivo inhibition of tumor growth to a tumor-bearing animal (in vivo by examining in), or, as will be described later, with respect to tumor cells expressing the polypeptide, investigate whether shows the cytotoxic activity via immune cells or complement in vitro (in vitro) it can be evaluated by.

[0015]

　Incidentally, each of the base sequence of a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2,4,6,8,10,12,14,16,18,20,22,24, SEQ ID NO: 1, 3, It has been shown to 5,7,9,11,13,15,17,19,21,23.

[0016]

　The amino acid sequence present invention is represented by SEQ ID NO: 4 in the Sequence Listing disclosed is the SEREX method using a canine testis tissue-derived cDNA library and leukemia canine patient serum, specifically in serum from cancer-bearing dogs polypeptides that bind with antibody present, also the amino acid sequence represented by SEQ ID NO: 2, as a human homologous factor (homologue), the amino acid sequence shown in SEQ ID NO: 6, as a cat homologous factor of SEQ ID NO: amino acid sequence is represented by 8 was isolated as a mouse homologous factor is the amino acid sequence of MRAP2 (see example 1 below).

[0017]

　In the present invention, among the MRAP2 proteins, antibodies that bind to portions expressed on the cell surface of cancer cells are preferably used. Specifically, the N-terminal portion of MRAP2 protein, SEQ ID NO: 10 (human), 14 (dog), 18 (cats) or 22 amino acid sequence represented by (mouse), or C-terminal part of the MRAP2 protein there, SEQ ID NO: 12 (human), 16 (dog), 20 (cats) or 24 polypeptide having the amino acid sequence represented by (mouse), or a fragment thereof (preferably, 7 or more consecutive their amino acid sequences of it consists of an amino acid), or these polypeptides and 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably an amino acid sequence with a sequence identity of 99% or more antibodies to the polypeptides are exemplified with, the antibodies of the invention bind to these polypeptides, and anti-tumor activity It includes all antibodies that exhibit.

[0018]

　Antibodies against the MRAP2 used in the present invention, as long as capable of exhibiting anti-tumor activity may be any type of antibody, for example, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies (e.g. bispecific antibody), a human antibody, or and the like humanized antibodies, chimeric antibodies, or single chain antibody (scFV). Antibodies used in the present invention may also antibody fragments such as Fab and F (ab ') 2 containing antigen binding fragments of such. These antibodies and fragments thereof, also can be prepared by methods known to those skilled in the art. In the present invention, to antibodies capable of specifically binding the MRAP2 protein or fragment thereof is desired, but is preferably a monoclonal antibody, as long as the stable production of homogenous antibodies, be polyclonal antibodies good. Further, when the subject is a human, it is desirable to avoid or suppress rejection reaction is a human or humanized antibody.

[0019]

　Here, the "MRAP2 protein or fragment that specifically binds the" specifically binds to MRAP2 protein or fragment thereof, means that does not substantially bind other proteins and.

[0020]

　Anti-tumor activity of antibodies that may be used in the present invention, by examining the inhibition of tumor growth to a tumor-bearing animals in vivo, or, as described below, to tumor cells expressing the polypeptide in vitro Te, can be evaluated by examining whether shows the cytotoxic activity via immune cells or complement.

[0021]

　Furthermore, the subject is a subject of treatment and / or prevention of cancer in the present invention include humans, companion animals, livestock, sports animals, a mammal, such as laboratory animals, the preferred subject is a human.

[0022]

　Hereinafter, production of antigens related to the present invention, the production of antibodies and the pharmaceutical compositions described.

[0023]

　
　protein or fragment thereof is used as the sensitizing antigen for obtaining antibodies against MRAP2 used in the present invention include humans, dogs, cats, mice, cows, horses, rats, chickens, etc. , animal species from which it is derived is not limited. But it is preferable to select in consideration of compatibility with the parent cell used for cell fusion, in general, preferably proteins from mammalian, especially human-derived protein is preferred. For example, MRAP2 cases of human MRAP2, or the like can be used cells expressing human MRAP2 protein or its partial polypeptide, a human MRAP2.

[0024]

　Nucleotide sequence and amino acid sequence of human MRAP2 and its homologues, for example, access the Web site of the GenBank (USA NCBI), BLAST, an algorithm such as FASTA (Karlin and Altschul, Proc Natl Acad Sci USA, 90:.... 5873 .. -5877,1993; Altschul et al, Nucleic Acids Res 25: 3389-3402, can be obtained by utilizing a 1997).

[0025]

　In the present invention, when relative to the amino acid sequence of the nucleotide sequences or SEQ ID NO: 2 of SEQ ID NO: 1 of human MRAP2, nucleotide sequence or amino acid sequence and 70% to 100% of these ORF or mature portion, preferably 80% 100%, more preferably 90% to 100%, more preferably 95% to 100%, for example 97% to 100%, 98% and 100% sequence identity of 99% to 100% or 99.5% and 100% nucleic acid or protein is the target consisting of a sequence having sex. Here, "% sequence identity" of two sequences without introducing or gaps or by introducing gaps, when aligned to maximize the similarity (alignment), the amino acid (or base) means the percentage (%) of identical amino acids (or bases) of the total number.

[0026]

　Fragments of MRAP2 protein, from amino acid long epitope antibodies is the smallest unit that recognizes (antigenic determinant) and has a length less than the full length of the protein. Epitopes mammal, preferably humans, refers to a polypeptide fragment having antigenicity or immunogenicity, its minimum unit, about 7 to 12 amino acids, for example, a 8-11 amino acids, the amino acid sequence of MRAP2 protein 80% or more, preferably 85% or more, more preferably 90% or more, more preferably include a polypeptide consisting of an amino acid sequence with a sequence identity of 95% or more.

[0027]

　Above, a polypeptide comprising a human MRAP2 protein or its partial peptide, for example, Fmoc method (fluorenyl methyloxy carbonyl method), be synthesized according to the chemical synthesis methods such as tBoc method (t-butyloxycarbonyl method) can (Japanese biochemical Society, biochemical experimental course 1, chemistry IV of the protein, the chemical modification and peptide synthesis, Tokyo Kagaku Dojin (Japan), 1981). It can also be synthesized by a conventional method using various commercially available peptide synthesizers. Further, known genetic engineering techniques (Sambrook et al, Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press, Ausubel et al, Short Protocols in Molecular Biology, 3rd edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), using a John Wiley & Sons, etc.), a polynucleotide encoding the polypeptide is prepared and introduced into the host cell incorporating the polynucleotide into an expression vector, said host Poripepu in a cell By producing de, can be obtained the desired polypeptide.

[0028]

　The polypeptide encoding polynucleotides, by a conventional method using a known genetic engineering technique or commercially available nucleic acid synthesizer, can be readily prepared. For example, DNA comprising the nucleotide sequence of SEQ ID NO: 1, PCR is performed using a pair of primers designed to the human chromosome DNA or cDNA library was used as a template, can be amplified nucleotide sequence described in SEQ ID NO: 1 it can be prepared by. The reaction conditions for PCR can be set appropriately, for example, thermostable DNA polymerase (e.g., Taq polymerase, etc.) and Mg 2+ using containing PCR buffer, 30 seconds at 94 ° C. (denaturation), and 30 seconds at 55 ° C. 1 min (annealing), as one cycle reaction process consisting of 1 minute (elongation) at 72 ° C., after for example 30 cycles, and the like can be mentioned conditions for reacting at 72 ° C. 7 minutes, but is not limited thereto. Method for PCR, for the conditions, eg, Ausubel et al, Short Protocols in Molecular Biology, 3rd edition, A compendium of Methods from Current Protocols in Molecular Biology (1995), are described in John Wiley & Sons (particularly Chapter 15) ing.

[0029]

　Further, based on the information of the nucleotide sequence and amino acid sequence represented by SEQ ID NO: 1-8 of the Sequence Listing herein, to prepare a suitable probe or primer, a cDNA library, such as a human using it by screening, it is possible to isolate the desired DNA. cDNA libraries, cells expressing the protein of SEQ ID NO: 2, 4, 6 or 8, it is preferably prepared from an organ or tissue. Examples of such cells and tissues, brain, leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma or anus it is a cell or tissue derived from a cancer or tumor such as ambient adenocarcinoma not limited thereto. Preparation of the probes or primers, construction of cDNA library, screening cDNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Sambrook et al, Molecular Cloning, 2nd edition, Current Protocols in Molecular Biology (1989), it can be performed according to the method described in Ausbel et al (above) and the like. From thus obtained DNA by, it is possible to obtain DNA encoding human MRAP2 protein or its partial peptide.

[0030]

　As the host cells, the long polypeptide and capable of expressing cells may be any one, such as E. coli Examples of prokaryotic cells, monkey kidney cells COS1 Examples of eukaryotic cells, Chinese hamster ovary cells mammalian cells such as CHO, human embryonic kidney cell line HEK293, mouse embryonic skin cell line NIH3T3, budding yeast, yeast cells such as fission yeast, silkworm cells, and the like Xenopus egg cells, but are not limited to.

[0031]

　When using a prokaryotic cell as a host cell, the expression vector preferably replicable origin in prokaryotic cells, a promoter, a ribosome binding site, multiple cloning site, a terminator, a drug resistant gene, auxotrophic complementary gene, a reporter gene using an expression vector having a like. Expression vectors for E. coli, pUC system, pBluescriptII, pET expression system and pGEX expression system. Incorporating DNA encoding the above polypeptide into such an expression vector, after a prokaryotic host cell transformed with the vector, followed by culturing the obtained transformant, prokaryotic polypeptides wherein said DNA encodes it can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.

[0032]

　When using eukaryotic cells as host cells, the expression vector, preferably a promoter, a splicing region, a eukaryotic expression vector having a poly (A) addition site and the like is used. Such expression vectors, pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag (A, B, C), such as pYES2 can be exemplified. Similar to the above, incorporating a DNA encoding the above polypeptide into such an expression vector After eukaryotic hosts cells with the vector and transformed by culturing the obtained transformant, the DNA encodes the by which polypeptides can be expressed in eukaryotic host cells. pIND / V5-His as an expression vector, in the case of using the pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like, His tag (e.g., (His) 6 ~ (His) 10), FLAG tag, myc tag , HA tag, as a fusion protein with the addition of various tags such as GFP, can be expressed the polypeptide.

[0033]

　Introduction into the host cells expression vectors, electroporation, calcium phosphate method, the liposome method, DEAE dextran method can be used microinjection, viral infection, lipofection, binding to a cell membrane permeable peptide, a known method etc. .

[0034]

　In order to isolate and purify a polypeptide of interest from the host cells may be carried out by combining known separation operations. The isolation and purification techniques, for example treatment with denaturing agents such as urea or a surfactant, ultrasonication, enzymatic digestion, salting-out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE , isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, but reverse phase chromatography, and the like, without limitation.

[0035]

　
　antibodies are usually heteromultimeric glycoproteins comprising at least two heavy chains and two light chains. Apart from IgM, antibodies of other classes, are two identical light (L) chains and two identical heavy (H) hetero tetrameric glycoproteins of about 150kDa composed of chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has intrachain disulfide bonds. Each heavy chain has a variable domain (VH region) at one end, followed by a number of constant regions. Each light chain has a variable domain (VL region), has one constant region at the end of the opposite. The constant region of the light chain is aligned with the first constant region of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Variable domain of an antibody specific regions confer binding specificity to the antibody indicate a particular variability called complementarity determining region (CDR). Relatively conserved portions of the variable region are called framework regions (FR). Variable domain of complete heavy and light chains comprise four FR connected by three CDR respectively. Three CDR is CDRH1 the heavy chain in this order from the N-terminal, CDRH2, CDRH3, are similarly called CDRL1, CDRL2, CDRL3 at light chain. The binding specificity of the antibody to the antigen, CDRH3 is most important. Also, CDR in each chain are held together in close proximity by the FR regions, which contributes to the formation of the antigen with CDR from the other strand. Constant regions do not directly contribute to antibody binding to an antigen, but exhibit various effector functions, such as participation of the antibody dependent cellular cytotoxicity (ADCC activity), phagocytosis via binding to Fcγ receptor, neonatal shown Fc receptor half-life / clearance rate via (FcRn), complement-dependent cytotoxicity activity via C1q component of the complement cascade (CDC activity).

[0036]

　
　The anti MRAP2 antibody in the present invention refers to an antibody having a full length or a fragment thereof immunologically reactive MRAP2 protein as described above.

[0037]

　Here, "immunological reactivity", antibody and MRAP2 antigen in or vitro vivo means the property of binding, fault tumor or tumor cells through such binding (e.g., killed , suppression or regression) functions, is exhibited. That is, antibodies used in the present invention, the tumor in combination with MRAP2 protein, preferably expressing MRAP2 protein on the cell surface (have) cancer, such as leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, if it is possible to disorders such as mast cell tumors or perianal gland cancer, not of any type.

[0038]

　Examples of antibodies include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies (e.g. bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, and single chain antibodies. Antibodies can also be, for example, antibody fragments (e.g., Fab or F (ab ') 2 comprising the antigen binding fragments of the like). The antibodies may be any class of immunoglobulin molecules, e.g. IgG, IgE, IgM, IgA, IgD and IgY or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 is such .

[0039]

　Antibodies Further, in addition to glycosylation, acetylation, formylation, amidation, phosphorylation, or pegs may be modified such as by (PEG) reduction.

[0040]

　The following illustrates the production of a variety of antibodies.

[0041]

　Polyclonal antibodies can be used in the present invention, for example, it can be obtained as follows.

[0042]

　Natural MRAP2 protein or recombinant MRAP2 proteins expressed in microorganisms such as Escherichia coli as a fusion protein, such as GST, or a partial peptide obtained mouse, human antibody-producing mice, small animals immunized serum of rabbits and the like. This, for example, be prepared by purifying by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column which was coupled MRAP2 proteins and synthetic peptides. In Examples described later, in the amino acid sequence of MRAP2 protein, mouse polyclonal antibodies are produced to the area expressed on the cell surface of cancer cells, the anti-tumor effect has been confirmed.

[0043]

　Another example of an antibody that can be used in the present invention is a monoclonal antibody. Monoclonal antibodies can, for example, can be obtained as follows. For example, immunized by administering cells (leukemia cell line K562 and malignant lymphoma cell line Namalwa, etc.) expressing MRAP2 to the cell surface mouse, extracting spleen from the mouse, on the cell separation, the cells and mouse fusing with myeloma cells, from among the resulting fused cells (hybridomas), selecting clones which produce antibodies with the cancer cell growth inhibitory action. The monoclonal antibody-producing hybridoma having cancer cell growth inhibitory action isolated, culturing the hybridoma, purification of the antibody by culture supernatant general affinity purification method, can be prepared.

[0044]

　Hybridomas producing monoclonal antibodies can be produced, for example be as follows. First of all, according to known methods, immunization of animals with a sensitizing antigen. As a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously in mammals. Specifically, it diluted sensitizing antigen in a suitable amount of PBS (Phosphate-Buffered Saline) or physiological saline with a conventional adjuvant optionally to that suspension, for example, Freund's complete adjuvant and after emulsification, It administered several times every 4 to 21 days in a mammal. It is also possible to use a suitable carrier at the time of immunization of the sensitizing antigen.

[0045]

　Thus immunizing mammals, after the desired antibody level is confirmation of the increase in serum, immunocytes are collected from the mammal and subjected to cell fusion, preferred immune cells, especially spleen cells and the like.

[0046]

　As the other parent cells to be fused with the above immune cells Mammalian myeloma cells are used. The myeloma cells are various known cell lines, for example, P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D.H. et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (deSt. Groth , S.F. et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S. J.Exp.Med. (1978) 148, 313-323), R210 Galfre, G. et al., Nature (1979) 277, 131-133) or the like is preferably used.

[0047]

　The cell fusion between the immune cells and the myeloma cells, known methods are basically, for example, Kohler and Milstein's method (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46 ) can be carried out in accordance with the like.

[0048]

　More particularly, the cell fusion is performed, for example, a cell fusion accelerator conventional nutrient broth in the presence of agents in. Examples of the fusion accelerator, for example, polyethylene glycol (PEG), is used Sendai virus (HVJ) or the like, may be added using an auxiliary agent such as dimethyl sulfoxide in order to increase the efficiency of the fusion desired.

[0049]

　The ratio of immunocytes to myeloma cells may be set arbitrarily. For example, preferably 1 to 10 times more immune cells than the myeloma cells. As the culture medium used for cell fusion, for example, a suitable RPMI1640 culture medium for the growth of the above myeloma cell lines, MEM culture medium, and other conventional culture medium used for this type of cell culture can be used, further , it can also be used in combination with serum supplements such as fetal calf serum (FCS).

[0050]

　Cell fusion, the mixed well with immune cells and the culture medium a predetermined amount of the myeloma cells, previously 37 ° C. of about the warmed PEG solution (e.g. having an average molecular weight of 1000 to 6000) 30 to 60% ( It was added at a concentration of w / v), to form a hybridoma of interest by mixing. Subsequently, sequential addition of a suitable culture liquid, to remove the cell fusion agents etc. which are undesirable for the growth of the hybridoma by repeating the operation of removing the supernatant by centrifugation.

[0051]

　Hybridomas obtained in this manner, the standard selection medium, are selected by culturing in example HAT medium (hypoxanthine, aminopterin, and culture medium containing thymidine). The cultivation in the HAT culture medium, the cells other than the desired hybridoma (non-fused cells) for a time sufficient to kill the (usually several days to several weeks). Then, the conventional limiting dilution method is performed to screen and single cloning of a hybridoma producing an antibody of interest.

[0052]

　In addition to obtaining the above hybridoma by immunizing non-human animals with antigens, human lymphocytes sensitized with e.g. protein human lymphocytes infected with EB virus in vitro, protein-expressing cells or their lysates, sensitization lymphocytes myeloma cells having a permanent division potential of human origin are fused with e.g. U266 (registration number TIB196), the desired activity (e.g., cell growth inhibitory activity) may be obtained hybridoma producing a human antibody with a.

[0053]

　Hybridomas producing monoclonal antibodies prepared in this way, it is possible to subcultured in a conventional culture medium, or can be stored for a long time in liquid nitrogen.

[0054]

　That is, the desired cells expressing the antigen or desired antigen as a sensitizing antigen and is immunized in the conventional method of immunization, known parent cells obtained immune cells by a conventional cell fusion method and fusion is allowed, by a conventional screening method, monoclonal antibody-producing cells (hybridomas) can be prepared by screening.

[0055]

　Here, the human antibody-producing mice, for example, KM mice (Kirin Pharma / Medarex) and Xeno mice (Amgen) is the known (e.g., International Publication No. WO02 / 02/43478 items, such as the No. WO02 / 092812). When immunized in such mice MRAP2 proteins or fragments thereof, complete human polyclonal antibodies can be obtained from the blood. Moreover, taking out spleen cells from mice after immunization, it is possible to generate fully human monoclonal antibodies by fusion method with myeloma cells.

[0056]

　Preparation of antigens, for example, a method using animal cells (JP-T 2007-530068) or a method using baculovirus (for example, International Publication No. WO98 / 46777) can be performed according to such. If the antigen has low immunogenicity, conjugated with macromolecules having immunogenic such as albumin, it may be carried out immunity.

[0057]

　Furthermore, an antibody gene cloned from a hybridoma, incorporating in a suitable vector, which is introduced into a host, can be used recombinant antibody produced using gene recombination technology (see, for example, Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, see Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA is synthesized in the variable region (V region) of the antibody using a reverse transcriptase from hybridoma mRNA. As long the DNA encoding the V region of an antibody of interest, it may be ligated to DNA encoding the desired antibody constant region (C region), which is then integrated into an expression vector. Alternatively, the DNA encoding the V region of the antibody may be integrated into an expression vector containing a DNA of an antibody C region. Expression control regions, for example, incorporated into an expression vector to express enhancer Under the control of the promoter. Subsequently, the expression vector transformed into a host cell and the antibody can be expressed.

[0058]

　Monoclonal antibodies are human monoclonal antibodies, non-human animal monoclonal antibodies (e.g., mouse monoclonal antibodies, rat monoclonal antibodies, rabbit monoclonal antibodies, chicken monoclonal antibody, etc.) and the like. Monoclonal antibodies, non-human mammal immunized with MRAP2 protein or fragment thereof (e.g., mouse, human antibody-producing mouse) can be produced by culturing a hybridoma obtained by fusion of spleen cells with myeloma cells from .

[0059]

　Chimeric antibodies are antibodies prepared by combining sequences derived from different animals, for example, the heavy chain of the mouse antibody, the heavy chain variable region and a human antibody light chain, is such as antibodies consisting of the constant region of the light chain . Preparation of the chimeric antibody can be carried out using known methods, for example, to connect the DNA encoding the DNA and human antibody C region encoding an antibody V region, and introduced into a host by incorporating this into an expression vector to produce the antibody.

[0060]

　The polyclonal antibody, human antibody-producing animals (e.g., mice) include antibodies obtained by immunizing MRAP2 proteins or fragments thereof.

[0061]

　Humanized antibodies are also referred modified antibody also reconstituted (reshaped) human antibody. Humanized antibodies, the CDR of an antibody derived from an immunized animal is constructed by transplanting into the complementarity determining region of a human antibody. The general gene recombination techniques are also known.

[0062]

　Specifically, the framework regions of the murine antibody CDR and a human antibody; several oligos were produced DNA sequence which was designed to connect the (framework region FR), so as to have a portion that overlaps the end portion It is synthesized by PCR method from the nucleotide. The resulting DNA was ligated with DNA encoding human antibody constant region, and then integrated into an expression vector, which is obtained by producing and introduced into a host (European Patent Application Publication No. EP 239 400, WO WO96 / reference No. 02576). FR of human antibody ligated through CDR, the complementarity determining region that forms a favorable antigen binding site is selected. If necessary, the complementarity determining region of reshaped human antibody may be replaced with amino acids in the framework region in the variable region of the antibody so as to form an appropriate antigen binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). Also, it may be substituted in the framework regions from different human antibodies (see International Publication No. WO99 / ​​51743).

[0063]

　After producing the chimeric antibodies or humanized antibodies, variable region (for example, FR) and the amino acid in the constant region may be substituted with other amino acids.

[0064]

　Amino acid substitutions, for example less than 15, less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or more than two amino acids, preferably 1 to 5 amino acids, more preferably 1 or 2 amino acids a substitution, substituted antibody should be functionally equivalent to the unsubstituted antibody. Substitutions, preferably conservative amino acid substitutions, which is the charge, side chains, polar, substitution between similar amino acids of the properties such as aromaticity. The similar amino acid properties, e.g., basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar sexual amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (threonine, valine, isoleucine), aromatic amino acids can be classified (phenylalanine, tyrosine, tryptophan, histidine) and the like.

[0065]

　Antibodies of the present invention may be modified antibodies. The modified antibodies, for example, a antibody bound to various molecules such as polyethylene glycol (PEG). In modified antibodies of the present invention, material to be bonded is not limited. To obtain such modified antibodies can be obtained by the obtained antibody chemically modified. These methods have already been established in the art.

[0066]

　Here, the phrase "functionally equivalent", the same biological or biochemical activity and antibody antibodies present invention of interest, in particular has a function of damaging tumors, human applications sometimes it refers to such that essentially cause rejection. Such activities include, for example, it can be exemplified cytostatic activity or binding activity.

[0067]

　A polypeptide functionally for preparing equivalent polypeptides, as the methods well known to those skilled in the art, methods of introducing mutations into polypeptides may be given. For example, the skilled artisan, site-directed mutagenesis (Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275, Zoller, MJ., And Smith, M. (1983) Methods Enzymol . 100, 468-500, Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456, Kramer, W. and Fritz, HJ., (1987) Methods Enzymol. 154, 350-367, Kunkel, TA., (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-27 6) by using a, by introducing an appropriate mutation into the antibody of the present invention you can be prepared functionally equivalent antibody antibody.

[0068]

　Antibodies that recognize the epitope of the anti MRAP2 antibody recognizes MRAP2 protein may be obtained by methods known to those skilled in the art. For example, an epitope conventional methods of anti MRAP2 antibody recognized MRAP2 proteins (e.g., epitope mapping, etc.) a method of determining by, to produce antibodies to a polypeptide having an amino acid sequence contained in the epitope as an immunogen, usually can epitopes of the produced antibodies was determined by the method, anti MRAP2 antibodies and epitope obtained by a method of selecting the same antibody. Here, "epitope" is a mammal, preferably in humans, refers to a polypeptide fragment having antigenicity or immunogenicity, its minimum unit, about 7 to 12 amino acids, preferably of 8-11 amino acids.

[0069]

　The affinity constant Ka of the antibody of the present invention (kon / koff) is preferably at least 10 7 M -1 , at least 10 8 M -1 , at least 5 × 10 8 M -1 , at least 10 9 M -1 , at least 5 × 10 9 M -1 , at least 10 10 M -1 , at least 5 × 10 10 M -1 , at least 10 11 M -1 , at least 5 × 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 is.

[0070]

　Antibodies of the present invention may be anti-tumor agents conjugated. The binding of the antibody to the anti-tumor agent, an amino group, a carboxyl group, hydroxy group, a thiol group and the reactive group (e.g., succinimidyl group, formyl group, 2-pyridyldithio group, Mareiimijiru group, an alkoxycarbonyl group it can be carried out via a spacer having such hydroxy group).

[0071]

　Examples of antitumor agents, known anti-tumor agents described below in literature, i.e., paclitaxel, doxorubicin, daunorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, thiotepa, busulfan, improsulfan, piposulfan, Benzodopa (Benzodopa ), carboquone, Metsuredopa (Meturedopa), Uredopa (Uredopa) Alto rate amine (Altretamine), triethylene melamine, triethylene phosphoramide, triethylene-thio phosphoramide (Tiaruaiethilenethiophosphoramide), trimethylol Loro melamine (Trimethylolomelamine), Buratashin, Buratashinon , camptothecin, bryostatin, kallistatin (c llystatin), cryptophycin 1, cryptophycin 8, dolastatin,'s Okaru clarithromycin, eleutherobin, punk Karachi statins, Sarkozy Kuching (sarcodictyin), sponge statins, chlorambucil, Kuroronafajin (chloRNAphazine), Korohosufamido (cholophosphamide), estramustine, ifosfamide, mechlorethamine, black Letter Min oxide hydrochloride, melphalan, Nobenbichin (novembichin), phenesterine (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard, carmustine, chlorozotocin (Chlorozoto in), fotemustine (fotemustine), lomustine, nimustine, ranimustine, calicheamicin (calicheamicin), Dainemaishin, clodronate, esperamicin, aclacinomycin, actinomycin, ose La clarithromycin (authramycin), azaserine, bleomycin, around actinomycin (cactinomycin), Karabishin (carabicin), carminomycin, Karujinofirin (carzinophilin), chromomycin, dactinomycin, Detorubishin (detorbicin), 6- diazo-5-oxo -L- norleucine, adriamycin (aDRIAMYCIN), epirubicin, esorubicin, idarubicin, Ma Cerro clarithromycin (marc Ellomycin), mitomycin C, mycophenolic acid (mycophenolic acid), Nogaramaishin (nogalamycin), Oribo mycin (olivomycins), peplomycin, Pot bafilomycin (potfiromycin), puromycin, Keramaishin (quelamycin), Rodorubishin (rodorubicin), streptonigrin , streptozocin, tubercidin (tubercidin), ubenimex, zinostatin (zinostatin), zorubicin (zorubicin), Denoputerin (denopterin), pteropterin (pteropterin), trimetrexate lysate (trimetrexate), fludarabine (fludarabine ), 6-mercaptopurine, Chiamipurin, thioguanine, ancitabine, azacitidine (azacitidine), 6- azauridine (Azauridine), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine (Enocitabine), floxuridine (Floxuridine); androgens, e.g. calusterone ( Calusterone), dromostanolone propionate, epitiostanol, mepitiostane, testolactone (testolactone), aminoglutethimide, mitotane, trilostane, Florin acid (frolinic acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine (amsacrine) , Best Love sill (bestrabucil), bisantrene (bisantrene), Edatorakiseto (edatraxate), Defofamin (defofamine), Demekorushin (demecolcine), Jiajikon (diaziquone), Eruforunichin (elfornithine), acetic acid Eripuchiniumu (elliptinium), epothilones (epothilone ), etoglucid (etoglucid), lentinan, lonidamine (lonidamine), maytansine (maytansine), ansamitocin (ansamitocine), mitoguazone (mitoguazone), mitoxantrone, Mopidanmoru (mopidanmol), Nitoraerin (nitraerine ), Pentostatin, Fenametto (Phenamet), pirarubicin, losoxantrone (Losoxantrone), podophyllin acid (podophyllinic acid), 2- ethyl hydrazide, procarbazine, Razokisan (Razoxane), rhizoxin, schizophyllan, spiro germanium (Spirogermanium), Tenyuazon acid (Tenuazonic acid), Toriajikon (triaziquone), roridins (roridine) A, Anguijin (anguidine), urethane, vindesine, dacarbazine, mannomustine (mannomustine), mitobronitol, mitolactol (mitolactol), pipobroman (pipobroman), Gashitoshin (ga ytosine), docetaxel, chlorambucil, gemcitabine (gemcitabine), 6- thioguanine, mercaptopurine, cisplatin, oxaliplatin, carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, Novantrone (novantrone), teniposide, edatrexate (edatrexate ), daunomycin, aminopterin, Kiseroda (Xeloda), ibandronate (ibandronate), irinotecan, topoisomerase inhibitors, difluoromethyl methylol two Chin (DMFO), retinoic acid, capecitabine (capecitabine), as well as their pharmaceutically acceptable salts or It encompasses those derivatives.

[0072]

　Alternatively, the antibodies of the present invention, known in the literature, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 SM, 212 Bi, 32 P, 175 Lu, 176 radioactive such as Lu it is also possible to combine the isotope. Radioactive isotopes are valid for tumor therapy and diagnosis is desirable.

[0073]

　Antibodies of the present invention, preferably, the antibody has a MRAP2 immunologically reactive, or an antibody specifically recognizing the MRAP2. Antibody, rejection should an antibody having a structure as little or no avoided in the subject animal to administer it. Such antibodies, for example, when the target animal is a human, a human antibody, a humanized antibody, a chimeric antibody (e.g. human - mouse chimeric antibodies), single chain antibodies, and the like bispecific antibodies. These antibodies, or variable regions of the heavy and light chains are of human origin antibodies, or the complementarity determining regions of the variable regions of the heavy and light chains from a non-human animal antibody (CDRl, CDR2 and CDR3 ) whether those consisting framework regions from human antibodies, or are those variable regions of the heavy and light chains are derived from a non-human animal antibody and the constant region of the heavy and light chain human antibody is a recombinant antibody is derived from. Preferred antibodies are the previous two antibodies.

[0074]

　These recombinant antibodies can be produced as follows. Monoclonal antibodies from antibody-producing cells such as hybridomas to human MRAP2 (e.g., human monoclonal antibodies, murine monoclonal antibodies, rat monoclonal antibodies, rabbit monoclonal antibodies, chicken monoclonal antibody, etc.) DNA encoding a cloned, which was a template the DNA encoding the light and heavy chain variable regions of an antibody produced by an RT-PCR method or the like, Kabat EU numbering system (Kabat et al, Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institute of Health , on the basis of Bethesda, in Md. (1991)) Te to determine the sequence or sequences of the CDR1, CDR2, CDR3 of the variable region of the light and heavy chains.

[0075]

　Furthermore, the DNA encoding the DNA or the CDR encoding each of these variable regions of a gene recombination technique (Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or by using a DNA synthesizer to produce. Here, the human monoclonal antibody-producing hybridomas, human antibody-producing animal (e.g., a mouse) after immunized with human MRAP2 to, can be produced by fusing the spleen cells with myeloma cells excised from the immunized animal . Alternatively, if desired, to produce DNA encoding the variable regions and constant regions of the light chain or the heavy chain derived from a human antibody using genetic recombination technique or a DNA synthesizer.

[0076]

　In the case of humanized antibody, a CDR coding sequences in the DNA encoding the variable region of the light or heavy chain from a human antibody, their corresponding non-human animal (e.g. mouse, rat, chicken, etc.) derived from CDR to produce a coding sequence and substituted DNA, respectively thereby resulting DNA, by ligating the DNA encoding the constant region of the light chain or the heavy chain derived from a human antibody, encoding the humanized antibodies of the antibody the to DNA can be prepared.

[0077]

　In the case of chimeric antibodies, non-human animal (e.g. mouse, rat, chicken, etc.) derived from an antibody of the light chain or DNA encoding the heavy chain variable region of each constant light or heavy chain from a human antibody by linking the DNA encoding the region, it is possible to produce a DNA encoding a chimeric antibody.

[0078]

　In the case of single-chain antibodies, the antibody is an antibody linked linearly through a linker and a heavy chain variable region and light chain variable regions encoding DNA, a linker encoding the heavy chain variable region DNA , and it is possible to create a DNA encoding the single chain antibody by binding the DNA encoding the light chain variable region. Here, both heavy and light chain variable regions are either of human origin antibodies, or, CDR only non-human animal (e.g. mouse, rat, chicken, etc.) is replaced by CDR of an antibody derived from of human origin antibodies. Further, the linker consists of 12-19 amino acids, for example of 15 amino acids (G4S) 3 (G -B Kim et al, Protein Engineering Design and Selection 2007, 20 (9):.. 425-432) and the like.

[0079]

　In the case of bispecific antibodies (diabody), the antibody are two different epitopes capable of specifically binding antibodies, encoding DNA, a light chain variable region B encoding for example the heavy chain variable region A DNA, DNA encoding the heavy chain variable region B, and to bind the DNA encoding the light chain variable region a in this order (however, the DNA encoding the DNA and heavy chain variable region B which encodes a light chain variable region B and is able to produce a DNA encoding a bispecific antibody by.) being coupled via a DNA encoding a linker as described above. Here, both heavy and light chain variable regions are either of human origin antibodies, or, CDR only non-human animal (e.g. mouse, rat, chicken, etc.) is replaced by CDR of an antibody derived from of human origin antibodies.

[0080]

　The recombinant DNA is prepared as described above, incorporated into one or more suitable vectors, which were introduced into a host cell (e.g., mammalian cells, yeast cells, insect cells, etc.), (co) expression is to be able to produce a recombinant antibody by (P.J. delves, aNTIBODY PRODUCTION ESSENTIAL TECHNIQUES, 1997 WILEY, P Shepherd and C. Dean, Monoclonal antibodies, 2000 OXFORD UNIVERSITY PRESS;..... J. W. Goding, Monoclonal Antibodies:.. principles and practice, 1993 ACADEMIC PRESS).

[0081]

　The antibody preferably has cytotoxic activity and thereby can exert an antitumor effect.

[0082]

　Further, to produce hybridomas capable of producing a different human antibody or non-human animal antibody (e.g. a mouse antibody) against human MRAP2, hybridomas were recovered monoclonal antibody produced, immunological binding and cytotoxic to human MRAP2 determines whether the object of antibody activity as an index. After thus identified a monoclonal antibody-producing hybridoma of interest, as described above, to prepare a DNA encoding the heavy chain and light chain variable regions of the antibody of interest from the hybridoma and sequenced, the DNA of another antibody utilized for the production.

[0083]

　Furthermore the antibody of the present invention so long as it has a specificity that specifically recognize MRAP2, in sequence and / or sequence of the constant region of the particular framework regions of the antibody, one or several (preferably, 1 or 2 Substitutes for an amino acid number), there may be deletions or additions. Here, several are 2 to 5, preferably means two or three.

[0084]

　The present invention further, DNA encoding the antibodies of the present invention or, DNA encoding the heavy chain or light chain of the antibody or also provides DNA encoding the heavy chain or the variable region of the light chain of the antibody .

[0085]

　Complementarity determining regions encoded by DNA of these sequences (CDR) are the regions that determine the specificity of the antibody, encoding the other regions (i.e., constant regions and framework regions) of the antibody sequence it may be sequences from other antibodies. Here is the other antibodies including antibodies derived from organisms other than human, preferably of human origin in terms of reducing side effects. That is, in the above-mentioned DNA, it is preferable that regions encoding framework regions and each constant region of the heavy and light chains contains a nucleotide sequence encoding the corresponding amino acid sequence derived from a human antibody.

[0086]

　DNA of the present invention, for example, can be obtained by the above method or the following method. First, from a hybridoma related to the antibody of the present invention, the total RNA was prepared using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers or the like. Then, in each of the variable regions of known mouse antibody heavy and light chain genes, by PCR using the sequences of the oligonucleotides are stored respectively in the primers to amplify the cDNA encoding the antibody. The sequence encoding the constant region, the known sequence can be obtained by amplified by PCR. Nucleotide sequence of DNA, by, for example, incorporated into a plasmid or phage for sequencing, it can be determined by a conventional method.

[0087]

　Antitumor effect against MRAP2 expressing cancer cells by anti MRAP2 antibody used in the present invention, the following mechanisms: MRAP2 expressing cells of effector cell antibody dependent cellular cytotoxicity (ADCC activity), or MRAP2 expression complement-dependent cell It believed to occur by cytotoxic activity (CDC activity).

[0088]

　Thus, the activity of anti MRAP2 antibody used in the present invention, as specifically shown in the Examples below, by measuring the ADCC activity or CDC activity against cancer cells expressing MRAP2 in vitro it can be evaluated.

[0089]

　Anti MRAP2 antibody used in the present invention is combined with MRAP2 protein on cancer cells, by the activity, because it exhibits an anti-tumor effect, may be useful in the treatment or prevention of cancer. That is, the present invention as an active ingredient an anti MRAP2 antibody provides a pharmaceutical composition for the treatment and / or prevention of cancer. When used for the purpose of administering the anti MRAP2 antibody to the human body (antibody treatment), in order to reduce immunogenicity, it is preferable that the human antibodies or humanized antibodies.

[0090]

　Incidentally, the higher the binding affinity between MRAP2 proteins on anti MRAP2 antibody and cancer cell surface by anti MRAP2 antibodies, anti-tumor activity is obtained stronger. Therefore, if acquired anti MRAP2 antibodies having MRAP2 protein and high binding affinity, it can be expected stronger antitumor effect, it is possible to adapt a pharmaceutical composition for the treatment and / or prevention of cancer. As high binding affinity, as described above, the binding constant (affinity constant) Ka (kon / koff) is preferably, 10 7 M -1 , at least 10 8 M -1 , at least 5 × 10 8 M -1 , at least 10 9 M -1 , at least 5 × 10 9 M -1 , at least 10 10 M -1 , at least 5 × 10 10 M -1 , at least 10 11 M -1 , at least 5 × 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 is desirably.

[0091]

　
　ability of the antibody to bind to MRAP2, such for example ELISA as described in Example, Western blotting, to identify by using a binding assay using such immunofluorescence and flow cytometry analysis be able to.

[0092]

　
　antibody that recognizes MRAP2 is by immunohistochemistry in a manner well known to those skilled in the art, tissue obtained from the patient during surgery, the patient's bone marrow tissue, lymph nodes, peripheral blood cells or bone marrow cells and, from obtained from animals bearing spontaneously or xenograft tissue inoculated with cell lines expressing MRAP2 after transfection tissue was fixed, paraffin-embedded frozen sections or paraformaldehyde and paraformaldehyde or acetone fixed tissue sections use can be tested for reactivity with MRAP2.

[0093]

　For immunohistochemical staining, an antibody that is immunologically reactive to MRAP2, can be dyed in a variety of ways. For example, by reacting horseradish peroxidase-conjugated goat anti-mouse antibody or goat anti-rabbit antibody can be visualized.

[0094]

　
　The present invention is an antibody of the present invention, i.e. antibodies or fragments thereof against MRAP2 described above (preferably an antigen-binding fragment) to provide a pharmaceutical composition comprising a (or pharmaceutical). The pharmaceutical compositions of the present invention (or pharmaceutical) usually contain antibodies or fragments thereof against MRAP2 described above the (preferably an antigen-binding fragment) in an effective amount.

[0095]

　Target of a pharmaceutical composition for the treatment and / or prevention of cancer of the present invention is not particularly limited as long as the MRAP2 gene (in the normal cell surface) expressing cancers (cells).

[0096]

　The term "tumor" and "cancer" as used herein, refers to a malignant neoplasm, are used interchangeably.

[0097]

　Cancer of interest in the present invention is a cancer expressing a gene encoding a polypeptide comprising a partial sequence thereof consisting of the amino acid sequence or 7 or more contiguous amino acids of SEQ ID NO: 2, 4, 6 or 8, preferably is a cancer that expresses such a polypeptide to the cell surface. Cancer of interest in the present invention are preferably leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, mastocytoma or perianal adenocarcinomas, these specific cancers, such as acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocyte hypercholesterolemia leukemia (leukocythemic leukemia), basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia , gross leukemia, leader cell leukemia, Schilling leukemia, stem cell leukemia, Ashiro blood leukemia, undifferentiated cell leukemia, hairy cell leukemia, blood cell sprouts Leukemia (hemoblastic leukemia), blood blasts leukemia (hemocytoblastic leukemia), histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenia leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphotropic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Negeri leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, non-Hodgkin's lymphoma (Burkitt's lymphoma (BL), small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL), follicular phosphorus Carcinoma (FL), diffuse large B-cell lymphoma (DLCL), marginal zone (marginal zone) lymphoma (MZL), hairy cell leukemia (HCL), lymphoplasmacytic leukemia (LPL), mucous membrane-binding compound lymphoid tissue ( extranodal marginal zone B-cell lymphoma of MALT), mediastinal large cell lymphoma, intravascular large cell lymphoma, primary effusion lymphoma, precursor B cell lymphoblastic leukemia / lymphoma, precursor T-cells and NK-cell lymphoma (precursor T cell lymphoblastic lymphoma, NK blast cell lymphoma), including tumor (peripheral T-cell lymphoma and leukemia (PTL) of mature T and NK cells), adult T-cell leukemia / T-cell lymphomas and large granular lymphocytic leukemia, T cell chronic lymphocytic leukemia / prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, nodules T-/ NK cell lymphoma, enteropathy type T-cell lymphoma, hepatic splenic T-cell lymphoma, non-structural large cell lymphoma (ALCL), angiography Se trick (angiocetric) and angioimmunoblastic T cell lymphoma, mycosis fungoides tumor / Sezary syndrome, cutaneous T-cell lymphoma (CTCL)), Hodgkin's lymphoma, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell lung cancer, small-cell lung cancer, glioma, astrocytes tumor, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumors, pineal blastoma, nuclear power plant sexual brain lymphoma, germ cell tumors, superficial colorectal cancer, tumor type colon cancer, ulcerative infiltrating colon cancer, diffuse infiltrating colorectal cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, superficial spreading melanoma , nodular melanoma, lentigo maligna melanoma, acral lentigo of black Melanoma, neuroblastoma, ganglion neuroblastoma, ganglion tumor, insulinoma, gastrinoma, glucagonoma, Bipoma, somatostatin-secreting tumor, carcinoid, pancreatic islet cell tumors, tumor type gastric cancer, ulcers localized type gastric cancer, ulcers infiltration-type gastric cancer, diffuse infiltrating gastric cancer, hepatocellular carcinoma and hepatoblastoma, epithelial ovarian carcinoma, borderline tumor, germ cell tumors, stromal tumors, serous adenocarcinoma, clear cell adenocarcinoma, endometrioid adenocarcinoma, transitional cell carcinoma, mucinous adenocarcinoma, mixed-ovarian cancer, squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma, Jinsengan, hypernephroma, renal fibrosarcoma, transitional cell cancer (renal pelvis and / or ureter (uterer)), mast cell tumors, perianal adenoma, although perianal adenocarcinoma are included, without limitation.

[0098]

　Moreover, animals of interest, is a mammal, for example a primate, pet animals, livestock, sports animals, a mammal including experimental animal, particularly a human, dogs and cats preferred.

[0099]

　When using the antibody used in the present invention as a pharmaceutical composition can be formulated by methods known to those skilled in the art. For example, a sterile solution with water or other pharmaceutically acceptable liquid, or in the form of suspensions of injection can be used parenterally. For example, carrier or medium or additives pharmacologically acceptable, specifically, sterilized water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles , preservatives, binding agents, and such, it is considered that formulated by mixing in a unit dosage form required for accepted pharmaceutical implementations. The active ingredient in the preparation is controlled in such a dose that an appropriate dose is obtained within the specified range given.

[0100]

　Sterile compositions for injection can be formulated following normal drug implementations using vehicles such as distilled water for injection.

[0101]

　Aqueous solutions for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents, for example D- sorbitol, D- mannose, D- mannitol, sodium chloride may be used in combination with an appropriate dissolution aid such as alcohol, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, non-ionic surfactants, such as polysorbate 80 (TM), may be used in combination with HCO-60.

[0102]

　Sesame The oily liquid, soybean oils, solubilizer such as benzyl benzoate, it may be used in combination with benzyl alcohol. Further, buffers such as phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, may be blended with the antioxidant. The prepared injection is generally filled into a suitable ampule.

[0103]

　Administration is oral or parenteral, preferably parenteral administration, in particular, injectable form, nasal dosage form, pulmonary dosage form, and percutaneous administration type. Examples of injectable dosage forms, for example, intravenous injection, intramuscular injection, intraperitoneal injection, can be administered systemically or locally by such as subcutaneous injection. The injection, infusion, the antibodies of the present invention can be administered directly into the tumor by topical administration to the tumor by embedding such a controlled release formulation.

[0104]

　Further, it is possible to select the patient's age, weight, sex, the appropriate administration method and the like symptoms. The dosage of the antibody or a pharmaceutical composition containing a polynucleotide encoding an antibody, for example, it is possible to choose the range of 1000mg from 0.0001mg per kg 1kg per administration. Alternatively, for example, although the dose can be selected in the range of 0.001 ~ 100000 / body per patient, not necessarily limited to these numerical values. The dosage, method of administration, patient's weight, age, sex, and condition, and can be appropriately selected by those skilled in the art.

[0105]

　Antibody or fragment thereof of the present invention, or the above-mentioned cancer by administering the pharmaceutical composition to a subject containing it, cancer expressing inter alia the MRAP2 the cell surface, preferably, leukemia, malignant lymphoma, lung cancer, brain tumors, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, kidney cancer, can be a mastocytoma or perianal adenocarcinoma treating and / or prophylaxis.

[0106]

　Further it includes pharmaceutical compositions of the present invention (or pharmaceutical), in combination with a pharmaceutical composition comprising an anti-tumor agent or anti-tumor agents as exemplified above (or pharmaceutical), administered in combination to a subject, cancer the method of treatment and / or prevention are also encompassed by the present invention. Cancer of interest is the same as described above. Antibody or fragment thereof and an antitumor agent of the present invention can be administered simultaneously or separately to the subject. When administered separately, it may be even or later be any pharmaceutical composition above, their administration interval, dose, administration route and frequency of administration may be chosen by the specialist. When administered simultaneously, for example, an antibody or fragment thereof and an antitumor agent of the present invention, a pharmaceutical composition of the pharmaceutical dosage form obtained by mixing formulated in a carrier (or medium) that are pharmacologically acceptable is also included and shall. Also, for any of the above pharmaceutical compositions and dosage forms containing anti-tumor agents, the formulation of the pharmaceutical compositions and dosage forms containing the antibodies of the present invention, formulation, route of administration, dose, such as cancer description can be applied.

[0107]

　Accordingly, the invention comprises administering the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising an anti-tumor agent as exemplified above, cancer treatment and / or a combination medicament for the prevention and it including also provides treatment and / or prevention of cancer. The present invention also provides for an antibody or fragment thereof and an antitumor agent of the present invention, together with carriers and / or additives pharmacologically acceptable, also a pharmaceutical composition for the treatment and / or prevention of cancer to.
Example

[0108]

　Hereinafter, although now be described by way of the present invention examples, the scope of the present invention is not intended to be limited these specific examples.

[0109]

　Example 1 SEREX Identification of Novel Cancer Antigen Protein by Method
　(1) Preparation of cDNA library
　acids from healthy canine testis tissue guanidinium - phenol - Total RNA was extracted by chloroform method (Acid guanidium-Phenol-Chloroform method), poly a RNA was purified according to the protocol attached to the kit using Oligotex-dT30 mRNA purification kit (Takara Shuzo).

[0110]

　Was synthesized dog testis cDNA phage library using the obtained mRNA (5 [mu] g). Using cDNA Synthesis Kit for preparing the cDNA phage library, ZAP-cDNA Synthesis Kit, a ZAP-cDNA GigapackIII Gold Clonig Kit ( STRATAGENE), to create a library in accordance with the protocol attached to the kit. The size of the cDNA phage library prepared in × 10 1 6 was pfu / ml.

[0111]

　(2) Screening of cDNA library with sera
　using canine testis cDNA phage library prepared above, immunoscreening was carried out. Specifically, were infected with phage host E. coli (XL1-Blue MRF ') such that about 2500 clones should appear on an NZY agarose plate Φ90 × 15mm, 42 ℃, 3 and incubated to 4 hours, the plaques (plaques) made allowed, IPTG (isopropyl-beta-D-thiogalactoside) penetration is allowed nitrocellulose membrane (Hybond C Extra: GE Healthcare Bio -Scinece) plates to induce and express proteins covering 4 hours at 37 ° C. with, the transfer of the protein to the membrane. Then the membrane was collected TBS containing 0.5% non-fat dry milk (10mM Tris-HCl, 150mM NaCl , pH7.5) was inhibited non-specific reaction by shaking overnight at immersed 4 ° C. to. This filter was allowed to react for 2-3 hours at room temperature and the 500-fold diluted canine patient serum.

[0112]

　As the above-described canine patient serum, it was used serum collected from canine patients suffering from leukemia. The serum was stored at -80 ℃, and pretreated immediately before use. Pretreatment method of serum, according to the following method. In other words, they were infected with lambda ZAP Express phage to which no foreign gene was inserted into a host E. coli (XL1-Blure MRF '), 37 ℃ on NZY plate medium and incubated overnight. Then 0.2 M NaHCO containing 0.5M NaCl 3 added buffer pH8.3 in the plate, 15 hours after standing at 4 ° C., the supernatant was collected as an E. coli / phage extract. Then, the recovered E. coli / phage extract NHS- was passed through a column (GE Healthcare Bio-Science), to immobilize proteins derived from the E. coli phage. This protein-immobilized column to canine patient serum was allowed to flow through and react remove antibodies adsorbed on E. coli and phage from the serum. The serum fraction that passed through the column was diluted 500-fold with TBS containing 0.5% non-fat dry milk, which was used as immunoscreening material.

[0113]

　After the membrane was blotted the thus treated serum and protein washed 4 times with TBS-T (0.05% Tween20 / TBS), 5000 -fold with TBS containing 0.5% non-fat dry milk as a secondary antibody was diluted goat anti-dog IgG (goat anti dog IgG-h + L HRP conjugated: BETHYL Laboratories) was added, reacted at room temperature for 1 hour, followed by detection by the enzyme coloring reaction using the NBT / BCIP reaction solution (Roche), color colonies that match seropositive sites taken from Φ90 × 15mm of NZY agarose plate, SM buffer (100 mM NaCl, 10 mM MgClSO 4 dissolved in, 50mM Tris-HCl, 0.01% gelatin pH 7.5) 500 [mu] l . In the same manner as described above until the color reaction positive colonies unifies, secondary, repeated tertiary screening, about 10,000 phage clones reactive with IgG in the serum was screened, a single one of the positive clones release was.

[0114]

　(3) Homology Search of Isolated Antigen Gene
　one positive clone isolated by the above methods for providing the nucleotide sequence analysis was carried out an operation to convert the phage vector to a plasmid vector. Specific to the host E. coli (XL1-Blue MRF ') absorbance OD 600 and solution 200μl which was prepared so as to be 1.0, purified phage solution 100 [mu] l, further ExAssist helper phage (STRATAGENE) 37 ℃ were mixed 1μl in 15 minutes after the reaction, of LB medium was 2.5 to 3 hours incubation at 3ml added 37 ° C., was incubated for 20 minutes at immediately 70 ° C. water bath, 4 ° C., then centrifuged 1000 × g, 15 min , and the supernatant was collected as a phagemid solution. Phagemid host E. coli (SOLR) absorbance OD then 600 and solution 200μl which was prepared to become 1.0, was mixed with phage solution 10μl purified, and reacted for 15 minutes at 37 ° C., 50 [mu] l ampicillin (final concentration 50 [mu] g / ml) and incubated overnight at 37 ° C. plated on LB agar medium containing. Taken single colony of SOLR was transformed, after incubation at 37 ° C. in ampicillin (final concentration 50 [mu] g / ml) containing LB medium, plasmid DNA was purified with an insert of interest using QIAGEN plasmid Miniprep Kit (QIAGEN) .

[0115]

　Purified plasmid using the T7 primer described in T3 primer SEQ ID NO: 26 as set forth in SEQ ID NO: 25, were analyzed insert the full-length sequence by a primer walking method. And obtaining a gene sequence described in SEQ ID NO: 3 The sequence analysis. Using a base sequence and amino acid sequence of this gene, the sequence was identical search against known genes perform sequence identity search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) the results, obtained gene was found to be MRAP2 gene. In human MRAP2 a human homologous factor of the canine MRAP2, sequence identity of nucleotide sequence 91%, 94% amino acid sequence, cat MRAP2 the sequence identity of nucleotide sequence 95%, the amino acid sequence 96%, mouse homologous in mice MRAP2 a factor, sequence identity of nucleotide sequence 84% were amino acid sequence 88%. The nucleotide sequence SEQ ID NO: 1 of human MRAP2, in the amino acid sequence SEQ ID NO: 2, the nucleotide sequence SEQ ID NO: 5 cats MRAP2, the amino acid sequence in SEQ ID NO: 6, the nucleotide sequence SEQ ID NO: 7 mice MRAP2, the amino acid sequence SEQ It is shown in the number 8.

[0116]

　(4) Gene expression analysis in each tissue
　dog to gene obtained by the above method, the human and mouse various normal tissues, various tumor tissues and RT-PCR expression in various cancer cell lines (Reverse Transcription-PCR) method It was examined by. Reverse transcription was carried out as follows. That is, each tissue 50 ~ 100 mg and each cell line 5 ~ 10 × 10 6 total RNA was extracted according to the attached protocol using cells from TRIZOL reagent (ThermoFisher Scientific). The cDNA was synthesized by following the protocol of the kit attached total RNA Superscript First-Strand Synthesis System for RT-PCR using (ThermoFisher Scientific). CDNA of human normal tissues (brain, testis, colon and placenta), Gene Pool cDNA (ThermoFisher Scientific), were used QUICK-Clone cDNA (Clontech) and Large-Insert cDNA Library (Clontech). PCR reactions obtained gene-specific primers (dog primers SEQ ID NO: 27 and 28, the human primers SEQ ID NO: 29 and 30, mice primers SEQ ID NO: 31 and 32) were carried out as follows using. That, cDNA samples 0.25μl prepared by reverse transcription reaction, the above primers each 2 [mu] M, of each dNTP 0.2 mM, the total volume added to the buffer attached to each reagent so that ExTaq polymerase 0.65u (Takara Shuzo) 25 [mu] l and then, using a Thermal Cycler (BIO RAD), 30 seconds at 94 ° C., 30 sec at 55 ° C., PCR was carried out with 30 cycles of 1 minute at 72 ° C.. As a result, as shown in FIG. 1, dogs MRAP2 gene in dogs tumor tissue mastocytoma, strong expression in perianal adenocarcinoma was observed (Figure 1). In humans MRAP2 gene expression in normal human tissues was not observed expression in most tissues (Fig. 2A). While in human cancer cell leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, strong expression of human MRAP2 gene in cell lines of renal cancer was observed (Figure 2B). Furthermore, mice MRAP2 gene, leukemia, melanoma, is expressed in cell lines neuroblastoma was detected (Figure 3).

[0117]

　Example 2 Human MRAP2 Preparation of protein
　(1) full-length cDNA encoding human MRAP2, and the extracellular region of human MRAP2 (hereinafter, hN-terminal MRAP2, hC terminus MRAP2) Cloning of cDNA encoding
　encodes human MRAP2 full-length cDNA is based on the gene of SEQ ID NO: 1 obtained in example 1, were cloned by the following method. PCR is, 1 [mu] l of the cDNA expression by RT-PCR method among various tissues and cells derived cDNA prepared was confirmed in Example 1, two kinds of primers having EcoRI and NotI restriction enzyme cleavage sequence (SEQ ID NO: 33 and 34 and each 0.4 .mu.M, 0.2 mM dNTPs, the total volume added to the buffer attached to each reagent so that the PrimeSTAR HS polymerase 1.25 U (Takara Shuzo) 50 [mu] l description) in, using a Thermal Cycler (BIO RAD), 10 seconds at 98 ° C., 15 sec at 55 ° C., was carried out by repeating 30 cycles of 1 minute at 72 ° C.. The above-described two kinds of primers were those which amplify the region encoding the entire amino acid sequence of SEQ ID NO: 2. After the PCR, the amplified DNA was electrophoresed on a 1% agarose gel to purify a DNA fragment of about 0.6kbp using QIAquick Gel Extraction Kit (QIAGEN). Amplification product obtained by PCR reactions described above was inserted into pcDNA3.1 (ThermoFisher Scientific) (hereinafter, human MRAP2 / pcDNA3.1). Further, by sequencing using a DNA sequencer, the amplification product was confirmed to be cDNA sequence encoding human MRAP2. The nucleotide sequence of the sequence human MRAP2 genes represented by SEQ ID NO: 1, sequence represented by SEQ ID NO: 2 shows the amino acid sequence of human MRAP2 protein.

[0118]

　Further, based on SEQ ID NO: 1, 2 kinds of primers each 0.4μM (described in SEQ ID NO: 35 and 36) containing a KpnI and EcoRI restriction enzyme cleavage sequences, 0.2 mM dNTPs, 1.25 U of PrimeSTAR HS polymerase ( the total amount of added buffer attached to each reagent so that Takara Shuzo) and 50 [mu] l, using a Thermal Cycler (BIO RAD), 10 seconds at 98 ° C., 15 sec at 55 ° C., 30 cycles of 30 seconds at 72 ° C. PCR was carried out by repeating. The above-described two kinds of primers of the sequences ID NO: 1, were those which amplify the region encoding MRAP2 amino acid sequence of the extracellular region of protein (N-terminal) (SEQ ID NO: 10). After the PCR, the amplified DNA was electrophoresed on a 1% agarose gel to purify a DNA fragment of about 130bp with the QIAquick Gel Extraction Kit (QIAGEN). Amplification product obtained by PCR reactions described above is connected to pSecTagB the cDNA encoding the mouse IgG2a Fc protein was inserted (ThermoFisher Scientific), human N-terminal MRAP2 extracellular region / mouse IgG2a Fc fusion protein (hereinafter, hN was an expression vector encoding the end side MRAP2-mIgG2aFc) (hereinafter, pSecB-hN-terminal MRAP2-mIgG2aFc). Further, by sequencing using a DNA sequencer, the amplification product was confirmed to be cDNA sequence encoding the hN-terminal MRAP2-mIgG2aFc. The nucleotide sequence is sequence represented by SEQ ID NO: 37 encoding the hN-terminal MRAP2-mIgG2aFc, sequence represented by SEQ ID NO: 38 shows the amino acid sequence of the hN-terminal MRAP2-mIgG2aFc.

[0119]

　Further, based on SEQ ID NO: 1, 2 kinds of primers (SEQ ID NO: 39 and described in 40) each 0.4μM containing KpnI and EcoRI restriction enzyme cleavage sequences, 0.2 mM dNTPs, 1.25 U of PrimeSTAR HS polymerase ( the total amount of added buffer attached to each reagent so that Takara Shuzo) and 50 [mu] l, using a Thermal Cycler (BIO RAD), 10 seconds at 98 ° C., 15 sec at 55 ° C., 30 cycles of 30 seconds at 72 ° C. PCR was carried out by repeating. The above-described two kinds of primers of the sequences ID NO: 1, were those which amplify the region encoding MRAP2 amino acid sequence of the extracellular region of protein (C-terminal) (SEQ ID NO: 12). After the PCR, the amplified DNA was electrophoresed on a 1% agarose gel to purify a DNA fragment of about 400bp with the QIAquick Gel Extraction Kit (QIAGEN). Amplification product obtained by PCR reactions described above is connected to pSecTagB the cDNA encoding the mouse IgG2a Fc protein was inserted (ThermoFisher Scientific), human C-terminal MRAP2 extracellular region / mouse IgG2a Fc fusion protein (hereinafter, hC was an expression vector encoding the end side MRAP2-mIgG2aFc) (hereinafter, pSecB-hC-terminal MRAP2-mIgG2aFc). Further, by sequencing using a DNA sequencer, the amplification product was confirmed to be cDNA sequence encoding hC terminal side MRAP2-mIgG2aFc. The nucleotide sequence is sequence represented by SEQ ID NO: 41 encoding hC terminal side MRAP2-mIgG2aFc, sequence represented by SEQ ID NO: 42 shows the amino acid sequence of hC-terminal MRAP2-mIgG2aFc.

[0120]

　(2) hN terminus MRAP2-mIgG2aFc Preparation
　was prepared hN-terminal MRAP2-mIgG2aFc for MRAP2 as an immunogen for making antibodies.

[0121]

　The expression vector PSecB-hN-terminal MRAP2-mIgG2aFc introduced by lipofection into human embryonic kidney cell line HEK293 cells was performed purification of hN-terminal MRAP2-mIgG2aFc from the culture supernatant after the introduction 7 days. The culture supernatant was applied to Hi Trap ProteinA HP column (GE Healthcare Bioscience), washed with binding buffer (20mM sodium phosphate (pH 7.0)), elution buffer (0.1M glycine--HCl (pH and eluted with 2.7)). The eluate was immediately neutralized by eluting into tubes plus neutralization buffer (1M Tris-HCl (pH 9.0)). Next, the buffer of the eluate obtained by the above method, using an ultrafiltration NANOSEP 10K OMEGA (PALL), was replaced physiological phosphate buffer (Nissui Pharmaceutical), HT Tuffryn Acrodisc 0. perform sterile filtration with 22μm (PALL), which was used in the following experiments.

[0122]

　(3) hC terminus MRAP2-mIgG2aFc Preparation
　was prepared hC terminus MRAP2-mIgG2aFc for MRAP2 as an immunogen for making antibodies.

[0123]

　The expression vector PSecB-hC-terminal MRAP2-mIgG2aFc introduced by lipofection into human embryonic kidney cell line HEK293 cells was performed purification of hC-terminal MRAP2-mIgG2aFc from the culture supernatant after the introduction 7 days. The culture supernatant was applied to Hi Trap ProteinA HP column (GE Healthcare Bioscience), washed with binding buffer (20mM sodium phosphate (pH 7.0)), elution buffer (0.1M glycine--HCl (pH and eluted with 2.7)). The eluate was immediately neutralized by eluting into tubes plus neutralization buffer (1M Tris-HCl (pH 9.0)). Next, the buffer of the eluate obtained by the above method, using an ultrafiltration NANOSEP 10K OMEGA (PALL), was replaced physiological phosphate buffer (Nissui Pharmaceutical), HT Tuffryn Acrodisc 0. perform sterile filtration with 22μm (PALL), which was used in the following experiments.

[0124]

　Example 3 Preparation of polyclonal antibody that binds to the extracellular region of MRAP2
　production of polyclonal antibodies to (1) N-terminal side MRAP2
　to obtain antibodies that bind to the N-terminal extracellular region of MRAP2, hN powder prepared above side MRAP2-mIgG2aFc 0.1mg as an antigen mixed with complete Freund's adjuvant (CFA) solution of equal volume, 4 times this every two weeks, were administered subcutaneously to mice. Then blood was collected to give the anti-serum containing the polyclonal antibody. Further, this antiserum was purified using a protein G carrier (GE Healthcare Bioscience), to obtain a polyclonal antibody against hN-terminal MRAP2-mIgG2aFc. Also, the mouse sera not administered the antigen was used as a control antibody that was purified using a protein G carrier in the same manner as described above.

[0125]

　(2) Preparation of polyclonal antibody against C-terminal MRAP2
　to obtain antibodies that bind to the C-terminal extracellular region of MRAP2, a hC terminus MRAP2-mIgG2aFc 0.1mg prepared above as an antigen, fully equal volume It was mixed with Freund's adjuvant (CFA) solution, 4 times this every two weeks, were administered subcutaneously to mice. Then blood was collected to give the anti-serum containing the polyclonal antibody. Further, this antiserum was purified using a protein G carrier (GE Healthcare Bioscience), to obtain a polyclonal antibody against hC terminus MRAP2-mIgG2aFc. Also, the mouse sera not administered the antigen was used as a control antibody that was purified using a protein G carrier in the same manner as described above.

[0126]

　(3) Establishment of the full-length human MRAP2 constant expressing cells
　Human MRAP2 / pcDNA3.1 prepared above were introduced by lipofection into CHO-K1 cells (ATCC), by selection by G418 (Nacalai) in 500 [mu] g / ml, the total length It was established CHO cell line expressing the human MRAP2 constantly (CHO- human MRAP2). Expression vectors cDNA encoding MRAP2 is not inserted (hereinafter, emp / pcDNA3.1) was used as a control cell cells were selected by introducing in the same manner as above (hereinafter, CHO-emp).

[0127]

　(4) Expression Analysis of antigen protein on the cell surface
　in MRAP2 specifically expressed on the established full-length human MRAP2 constant expressing cell surface (3), to determine whether the reaction is a polyclonal antibody prepared in (1) It was. CHO- human MRAP2 cells and CHO-emp cells, respectively 10 6 to 6 cells were centrifuged at 1.5ml microcentrifuge tube. To this was added the above (1) polyclonal antibody 2 [mu] g (5 [mu] l) to the N-terminal MRAP2 prepared in after a further suspended in PBS containing 0.1% fetal bovine serum 95 [mu] l, and allowed to stand on ice for 1 hour. After washing with PBS, suspended in PBS containing 5μl of FITC-labeled goat anti-mouse IgG antibody (Santa Cruz) and 0.1% fetal bovine serum 95 [mu] l (FBS), and allowed to stand on ice for 1 hour. After washing with PBS, fluorescence intensity was measured with FACS Calibur Becton Dickinson and Company. On the other hand, similar to the above operation was performed using the control antibody prepared in the above (1) instead of the polyclonal antibody against MRAP2, was used as a control. As a result, the CHO- human MRAP2 cells added anti-N-terminal side MRAP2 polyclonal antibody, compared to the control, it showed enhanced 215% of the fluorescence intensity. On the other hand, the same procedure was performed on CHO-emp cells. As a result, CHO-emp cells added anti-N-terminal side MRAP2 polyclonal antibody, compared to the control, showed enhanced 0% of the fluorescence intensity. Therefore, the anti-N-terminal side MRAP2 polyclonal antibody was found to bind to MRAP2 proteins specifically expressed on the cell membrane surface. Incidentally, enhancement ratio of the fluorescence intensity is expressed by the rate of increase in mean fluorescence intensity in each cell (MFI value), was calculated by the following equation.

[0128]

　The average rate of increase in fluorescence intensity (enhancement ratio of fluorescence intensity) (%) = ((MFI value of cells reacted with anti-human MRAP2 antibody) - (control MFI value)) ÷ (control MFI value) × 100.

[0129]

　Next, (3) MRAP2 specifically expressed on the established full-length human MRAP2 constitutively expressed cell surface in examined whether the reaction is a polyclonal antibody prepared in (2). CHO- human MRAP2 cells and CHO-emp cells, respectively 10 6 to 6 cells were centrifuged at 1.5ml microcentrifuge tube. To this was added the above (2) polyclonal antibody 2 [mu] g (5 [mu] l) for C-terminal MRAP2 prepared in after a further suspended in PBS containing 0.1% fetal bovine serum 95 [mu] l, and allowed to stand on ice for 1 hour. After washing with PBS, suspended in PBS containing 5μl of FITC-labeled goat anti-mouse IgG antibody (Santa Cruz) and 0.1% fetal bovine serum 95 [mu] l (FBS), and allowed to stand on ice for 1 hour. After washing with PBS, fluorescence intensity was measured with FACS Calibur Becton Dickinson and Company. On the other hand, similar to the above operation is performed using the control antibody prepared in (2) above instead of the polyclonal antibody against MRAP2, was used as a control. As a result, the CHO- human MRAP2 cells added anti C-terminal MRAP2 polyclonal antibody, compared to the control, showed enhanced 223% of the fluorescence intensity. On the other hand, the same procedure was performed on CHO-emp cells. As a result, CHO-emp cells added anti C-terminal MRAP2 polyclonal antibody, compared to the control, showed enhanced 0% of the fluorescence intensity. Therefore, the anti-C-terminal MRAP2 polyclonal antibody, revealed that MRAP2 to protein specifically binds expressed on the cell membrane surface. Incidentally, enhancement ratio of the fluorescence intensity is expressed by the rate of increase in mean fluorescence intensity in each cell (MFI value), was calculated by the following equation.

[0130]

　The average rate of increase in fluorescence intensity (enhancement ratio of fluorescence intensity) (%) = ((MFI value of cells reacted with anti-human MRAP2 antibody) - (control MFI value)) ÷ (control MFI value) × 100.

[0131]

　Next, MRAP2 expression of the gene many confirmed leukemia cell line two (K562, THP-1), for malignant lymphoma cell lines (Namalwa), we investigated whether MRAP2 protein on its cell surface is expressed . Each human cell lines 10 each gene expression was observed in the 6 to 6 cells were centrifuged at 1.5ml microcentrifuge tube. To this was added the above (2) polyclonal antibody 2 [mu] g (5 [mu] l) for C-terminal MRAP2 prepared in after a further suspended in PBS containing 0.1% fetal bovine serum 95 [mu] l, and allowed to stand on ice for 1 hour. After washing with PBS, suspended in PBS containing 5μl of FITC-labeled goat anti-mouse IgG antibody (Santa Cruz) and 0.1% fetal bovine serum 95 [mu] l (FBS), and allowed to stand on ice for 1 hour. After washing with PBS, fluorescence intensity was measured with FACS Calibur Becton Dickinson and Company. On the other hand, similar to the above operation is performed using the control antibody prepared in (2) above instead of the polyclonal antibody against C-terminal MRAP2, was used as a control. As a result, the cells added anti C-terminal MRAP2 polyclonal antibody, compared to the control, were both strong fluorescence intensity is 30% or more. Specifically, K562 is 197%, THP-1 is 123%, Namalwa showed enhanced 104% of the fluorescence intensity. Therefore, it was confirmed that MRAP2 protein on the cell membrane surface of the human cancer cell lines is expressed. Incidentally, enhancement ratio of the fluorescence intensity is expressed by the rate of increase in mean fluorescence intensity in each cell (MFI value), was calculated by the following equation.

[0132]

　The average rate of increase in fluorescence intensity (enhancement ratio of fluorescence intensity) (%) = ((MFI value of cells reacted with anti-human MRAP2 antibody) - (control MFI value)) ÷ (control MFI value) × 100.

[0133]

　Example 4 Antitumor effect on cancer cells of a polyclonal antibody against MRAP2 (ADCC activity)
　then polyclonal antibodies against MRAP2 was investigated whether it is possible to impair tumor cells expressing MRAP2. It was evaluated using a polyclonal antibody against human N-terminal MRAP2 or human C-terminal MRAP2 prepared in Example 3. Human leukemia cell line K562 which expression is confirmed in MRAP2, the human malignant lymphoma cell line Namalwa 10 6 collected into centrifuge tubes or 50ml ml and incubated for 2 hours at 37 ° C. was added chromium 51 100 .mu.Ci. Then washed three times with RPMI1640 medium containing 10% fetal bovine serum, 10 per 96-well V-bottom plate 1 well 3 was added in pieces. Thereto, each polyclonal antibody against the human MRAP2 added 1 [mu] g, still mice were isolated from peripheral blood lymphocytes 2 × 10 each 5 were added one by one, 37 ° C., 5% CO 2 4 hours under the conditions of and cultured. After incubation, measuring the amount of chromium (Cr) 51 in the culture supernatant released from tumor cells injured, the ADCC activity against cancer cells by a polyclonal antibody against human N-terminal MRAP2 and human C-terminal MRAP2 calculated. As a result, with respect to K562 cells, 15.7%, respectively, 16.3%, relative to Namalwa cells, 14.8% respectively, 14.0% of the ADCC activity was confirmed (see Figure 4). On the other hand, for each cell line, when the same operation was carried out using the control antibody prepared from peripheral blood of mice not immunized (Example 3) with the antigen, and in the case of not adding the antibody , the activity was hardly observed (see Fig. 4). Thus, the ADCC activity using an antibody against MRAP2, revealed that it is possible to impair tumor cells expressing MRAP2.

[0134]

　Incidentally, cytotoxic activity in FIG. 4 (ADCC activity), as described above, antibodies against MRAP2 used in the present invention, 10 were incorporated mouse lymphocytes and chromium 51 3 the cells lines were mixed with 4 and culture time, by measuring the amount of chromium 51 released into the medium after the culture, the results showing cytotoxic activity against leukemia cell lines, which is calculated by the following equation *.

[0135]

　* Formula: Cytotoxic activity (%) = MRAP2 chromium 51 release amount × 100 from chromium 51 release amount ÷ 1N hydrochloric acid target cells were added from K562 when added antibody and mouse lymphocytes against.

Industrial Applicability

[0136]

　Antibodies of the present invention are useful for the treatment and / or prevention of cancer.

[0137]

　All publications cited herein, patents and patent applications are intended to directly incorporated herein by reference.

The scope of the claims

[Requested item 1]

　SEQ ID NO: 2, 4, 6 or 8 amino acid sequence represented by, or a fragment containing the MRAP2 protein, or 7 or more contiguous amino acids having an amino acid sequence, having the amino acid sequence 80% or more sequence identity to , to include as the antibody or active ingredients a fragment thereof having an immunological reactivity, a pharmaceutical composition for the treatment and / or prevention of cancer.

[Requested item 2]

　The MRAP2 protein a partial polypeptide, a polypeptide consisting of not less than 7 consecutive amino acids of the amino acid sequence represented by any one of the even sequence number among the sequence numbers 10 to 24 or the amino acid sequence and 80, including% or more sequence identity polypeptide consisting of an amino acid sequence having and an antibody or fragment thereof having an immunological reactivity as an active ingredient, a pharmaceutical composition according to claim 1.

[Requested item 3]

　Wherein the cancer is a cancer expressing MRAP2 on the cell surface The pharmaceutical composition according to claim 1 or 2.

[Requested item 4]

　Wherein the cancer is leukemia, malignant lymphoma, lung cancer, brain cancer, colon cancer, melanoma, neuroblastoma, pancreatic cancer, stomach cancer, liver cancer, ovarian cancer, esophageal cancer, renal cancer, selected from the group consisting of mast cell tumors and perianal adenocarcinoma that the pharmaceutical composition according to any one of claims 1 to 3.

[Requested item 5]

　Wherein the antibody is a monoclonal or polyclonal antibody, a pharmaceutical composition according to any one of claims 1-4.

[Requested item 6]

　Wherein said antibody, human antibody, humanized antibody, chimeric antibody, a single chain antibody or multispecific antibody, a pharmaceutical composition according to any one of claims 1 to 5.

[Requested item 7]

　A N-terminal partial polypeptide of MRAP2 protein, amino acid sequence represented by SEQ ID NO: 10, 14, and 18 or 22, or an amino acid sequence having the amino acid sequence 80% or more sequence identity, with the polypeptide consisting of antibody or fragment thereof having immunological reactivity.

[Requested item 8]

　A C-terminal partial polypeptide of MRAP2 protein, amino acid sequence represented by SEQ ID NO: 12, 16, 20 or 24, or an amino acid sequence having the amino acid sequence 80% or more sequence identity, with the polypeptide consisting of antibody or fragment thereof having immunological reactivity.

[Requested item 9]

　Wherein said antibody, human antibody, humanized antibody, chimeric antibody, a single chain antibody or multispecific antibody, antibody or fragment thereof according to claim 7 or 8.

[Requested item 10]

　A pharmaceutical composition according to any one of claims 1 to 6, comprising a pharmaceutical composition comprising an anti-tumor agent, a combination medicament for the treatment and / or prevention of cancer.

[請求項11]

　SEQ ID NO: 2, 4, 6 or 8 amino acid sequence represented by, or a fragment containing the MRAP2 protein, or 7 or more contiguous amino acids having an amino acid sequence, having the amino acid sequence 80% or more sequence identity to , and the antibody or fragment thereof having immunological reactivity comprising administering to a subject, the treatment and / or prevention of cancer.