JP2001286284A - Agent for genetically diagnosing and/or treating tumor using tumor-specific antigen and new application of proton pump-inhibitor as antitumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antitumor agent as a new application of a pharmaceutical composition or a proton pump-inhibitor [V-ATPase inhibitor, H+/K+-ATPase inhibitor, V-ATPase uncoupler (containing H+/Cl- symporter)] containing, as an active ingredient, tumor-specific antigen peptide, DNA and RNA encoding the peptide, antibody against the peptide, recombinant vector containing the DNA or RNA and transformant containing the recombinant vector, DNA or RNA hybridizing to the DNA or RNA under a stringent condition (antisense DNA or antisense RNA), or leucocyte reacting with the peptide or binding with an antibody against the protein. SOLUTION: A tumor-specific antigen peptide that contains at least an amino acid sequence constituting c", c or a subunit of V-ATPase, and tumor-specifically expresses, an antibody against the peptide, and an agent for treatment or diagnosis using DNA or RNA encoding the peptide, as well as a new application of a proton pump-inhibitor as an antitumor agent are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a tumor-specific antigen peptide, DNA and RNA encoding the peptide, a pharmaceutical composition containing an antibody against the peptide, and a novel application of a proton pump inhibitor as an antitumor therapeutic agent.

[0002]

2. Description of the Related Art Cancer treatments include surgery, chemotherapy, radiation therapy and immunotherapy. Among them, cancer-specific immunotherapy has recently attracted attention. As the genetic changes at the molecular level of cancer cells are revealed, it has been shown that cancer cell membranes have a different structure from normal membranes. This immunologically means that the antigen recognized by the immune cells is present on the cancer cell membrane. One of the antigens is
It is an antigen recognized by T cells and is called a tumor rejection antigen expressed in a form bound to its own MHC. The other is a tumor antigen on the cancer cell membrane recognized by NK cells and macrophages,
It is considered to be expressed without combining with C. Regarding the former tumor rejection antigen, in recent years, a gene encoding a tumor rejection antigen has been isolated from human melanoma, and more antigens have been found. As such a human tumor rejection antigen, for example, the epitope sequence “EA
DPTGHSY ”(referred to as MAGE-1), epitope sequence“ AARAVFLA
L '' (referred to as BAGE), the epitope sequence `` YRPRPRRY '' (GAGE-1
Epitope sequence `` VLPDVFIRC '' (NA17-A)
MAGE-1 is not expressed in normal tissues except for testis, and is considered to exhibit high tumor specificity. The expression of NA17-A is observed in about 50% of melanomas, and is slightly expressed in brain tumors and sarcomas, but is slightly expressed in normal tissues. In addition, melanocyte-specific tissue differentiation antigens or tumor-specific antigens derived from genetic changes associated with canceration have been identified (Robbins P
F etal., Curr. Opin. Immunol. 8: 628-638, 1996 / Hideyuki Ikeda., Annual Review Immunology 1996: 176-183, 1996./Kyogo Ito, Shigeki Nanashiki., History of Medicine 179: 117- 121, 199
6.)

[0003] Recently, trials of cancer-specific immunotherapy in which these tumor rejection antigen peptides are administered as vaccines to cancer patients have begun, and effective results have been reported (M
archand M et al., Int. J. Cancer. 63: 883-885, 199
5; Gjertsen MK et al., Int. J. Cancer. 72: 784-9
0, 1997; Schmidt W et al., Proc. Natl. Acad. Sci.
1: 3262-3267, 1997; Ben-Yedidia T et al., Curr. Op
in.Biotechnol. 8: 442-448, 1997; Nair SK et al.,
Eur. J. Immunol. 27: 589-597, 1997; Guo Yet al.,
Nature. Medicine. 3: 451-455, 1997).

Further, purified plasmid DNA (= naked DN)
It was reported that by dissolving A) in a 20% aqueous sucrose solution and injecting it directly into the thigh muscle of mice, the desired gene integrated on the plasmid could be expressed near the injection site (Wolff J Aet). al., Science. 247: 146
5-1468, 1990), research on gene therapy for cancer using direct DNA injection (plasmid DNA vaccine) has been actively conducted.

[0005] In recent years, a series of studies for the development of antisense drugs for cancer have started. Basically, the majority relate to S-oligomers (phosphorotioate oligomers), but also D-oligomers (phosphodiester oligomers), M-oligomers (methyl phosphonate oligomers), diS-oligomers (dithiophosphorotiate oligomers) There are those that apply the above. The mechanism of action of antisense oligomers is considered to be centered on the degradation of RNaseH, which is abundant in the nucleus of cells and hybrids of target mRNA and antisense oligomers (Wagner
R W. Nature. 372: 333-335, 1994). Furthermore, DNA can be used to inhibit translation of mRNA for the same purpose as antisense RNA. For example, a copy of a gene may be
on) and transferred to the host cell, the host cell
An antisense strand is created that is complementary to A, which complements the mRNA and inhibits protein translation. Thus, attempts to use antisense DNA as a drug (antisense agent) have begun.

Thus, there are many therapeutic options for tumor-specific antigens, but most importantly, to identify tumor-specific antigens that are commonly overexpressed on many types of cancer cell membranes. is there. However, to date, no such antigen has been found.

In general, the proton pump in the medical field often refers to a proton pump H + / K + -ATPase related to gastric acid secretion. H + / K + -ATPase inhibitors (commonly known as proton pump inhibitors; PPIs) are widely used worldwide for the treatment of gastric ulcers and duodenal ulcers, and have a strong acid secretion inhibitory effect of PPI and Helicobacter pylori (H. pyl).
The treatment of ulcers has been transformed by eradication therapy of ori). However, there are various other proton pumps. The so-called P-type ATP-dependent proton pump (H + -transporting ATPase), which forms a phosphorylated intermediate,
In addition to K + -ATPase, H +-present on plant and yeast cell membranes
ATPase is known, and its main function is to lower extracellular pH by releasing extracellular protons. Unlike these P-type ATPases (P-ATPases), H + -transporting ATPases that do not produce phosphorylated intermediates
FoF1-AT, which is present in the inner mitochondrial membrane and bacterial cell membrane, and has ATP synthesis as its main function
Pase is typical, and is known as F-type ATPase (F-ATPase). H + -transporting ATP very similar to this F-ATPase
Vacuolar H + -ATPase (vacuolar H + -ATPase; V
-ATPase) was mainly found on the intracellular granules of eukaryotic cells (Forgac M., Physiol. Rev. 69: 765-795, 1989). V
-ATPase is very similar in basic structure to F-ATPase (Forgac M., Physiol. Rev. 69: 765-795,
1989; Yoshinori Moriyama, Biochemistry 65: 413-436, 1993). That is, it is a multi-subunit enzyme composed of 9-11 subunits and is insensitive to vanadate like F-ATPase.
The cDNA for subunit c, a 16 kDa proteolipid, has already been isolated and sequenced (Nezu J et al., J.
Biochem. (Tokyo) 112: 212-219, 1992). This protein is an extremely hydrophobic protein with four transmembrane domains. It functions not only as a proton channel but also as a gap junction (Finbow M et al., J. Cell. Sci.
107: 1817-1824, 1994) and in some cases, the acetylcholine receptor (Birman S et al., FEBS. Lett. 261: 30
3-306, 1990).

In addition to the above-mentioned proton pumps, there are also a respiratory chain proton pump and a light-driven proton pump such as rhodopsin. Although there are many types and types of proton pumps, it is clear that V-ATPase plays an important role in the expression of biological activity via vacuolar acidic granules such as lysosomes and Golgi apparatus. Is becoming
Research has been focused on elucidating its function. V-ATPa
So many V-ATs to elucidate the function of se
Pase inhibitors have been developed and reported, but only one type of bafilomycin has been associated with cancer. That is, bafilomycin has a differentiation-inducing effect, a cell growth-inhibiting effect, and a tumor growth-suppressing effect on pancreatic cancer (Ohkuma S et
al., In. vitro. Cell. Develop. Biol. 29: 862-866,
1993; Ohta Tet al., Brit. J. Cancer. 73: 1511-151.
7, 1996; Ohta T et al., Gan ToKagaku Ryoho. 23: 16
60-1664, 1996), patent-related anticancer activity (US Pat. No. 5,324,720), cancer metastasis inhibitor (JP-A-9-301991), and anti-cancer drug enhancer (JP-A-10-120562). Can be seen. On the other hand, H + / K + -ATPase inhibitors are also attracting attention because they are related to Helicobacter pylori and gastric cancer development.However, no studies have been conducted on the anticancer effects of H + / K + -ATPase inhibitors. Recently, it has been reported that lansoprazole, one of the H + / K + -ATPase inhibitors, has an anti-adhesion effect on the extracellular matrix of cancer cells (Ohta T et al., Int. J. Oncol.
15: 33-39, 1999).

[0009] H + / Cl- symporter
Is a compound [ionophore] / transporter that has the activity of transporting H + and Cl- simultaneously, but the compound having such activity uncouples the pump (without inhibiting ATP hydrolysis activity, , Inhibiting proton transport activity). In that sense, it can be said that the H + / Cl- symporter is similar to an inhibitor for V-ATPase as an enzyme protein. The H + / Cl- symporter is a calcium antagonist, bepridil (Bepridil, 1-Isobutoxy-2-pyrr)
olidino-3- [N-benzylanilino] propane) C24H34N2O; MW =
366.54 and manganese tetraphenylporphine (5,10,1
5,20-Tetraphenyl-21H, 23H-porphine manganese (III) C
hloride), thallium chloride (TlCl ₃ ), and many compounds such as cycloprodigiosin have been reported. However, among them, BE-18591 (C22H35N3O.HCl) (Kojiri K et al.,
J. Antibiot. 46: 1799-1803, 1993; Nakajima S et a
l., J. Antibiot. 46: 1894-1896, 1993).
Regarding patents, a description is given of the effect of cycloprodigiosin on enhancing an anticancer drug (Japanese Patent Application Laid-Open No. 10-120562), but the antitumor effect of cycloprodigiosin itself has not been studied at all.

It is generally believed that the above-mentioned anticancer effect is only one of the various pharmacological effects possessed by the compounds bafilomycin, lansoprazole and BE-18591. That is, each of these three drugs is simply V-ATP
It only belongs to ase inhibitor group, H + / K + -ATPase inhibitor group, V-ATPase uncoupling agent or H + / Cl-symporter group and inhibits V-ATPase and H + / K + -ATPase To do
It has not been pointed out that V-ATPase uncoupler or H + / Cl- symporter is closely related to anticancer activity. It is V-ATPase, V-ATPase uncoupler or H + / K +
-This is due to the lack of an idea linking ATPase and H + / Cl- symporter to cancer and the lack of scientific evidence to support it. Evidence suggests that many V-ATPase inhibitors and H + / K
+ -ATPase inhibitors, V-ATPase uncouplers or H + / Cl- symporters have been discovered, and some of the drugs are widely used in the world. At the moment, no attention has been paid at all. That is, V-ATPase inhibitor, H + / K +-
The scientific notion that proton pump inhibitors, including ATPase inhibitors and V-ATPase uncouplers or H + / Cl- symporters, are potent antitumor agents has not been established so far.

[0011]

The present invention relates to a tumor-specific antigen peptide, DNA and RNA encoding the peptide, an antibody against the peptide, a recombinant vector containing the DNA or RNA, and a trait containing the recombinant vector. Transformant, the DNA
Alternatively, DNA or RNA (antisense DNA or antisense RN) that hybridizes with RNA under stringent conditions
A) or a pharmaceutical composition comprising an active ingredient comprising leukocytes which react with the peptide or leukocytes which bind to an antibody against the peptide, and a proton pump inhibitor [V-ATPase inhibitory action, H
+ / K + -ATPase inhibitory action, V-ATPase uncoupling agent (including H + / Cl-symporter)] as an antitumor agent as a novel use.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, extracted antigens from cell membrane components of a cell line established from human thyroid cancer, and obtained tumor-specific antigens from the antigens. The peptide was isolated, the partial nucleotide sequence was determined first, and then the entire nucleotide sequence was successfully determined. Further, Nishigori et al. In 1998 reported the tumor-specific antigen.
Proteolipid subunit of V-ATPase reported in June (ATP6F; equivalent to the 21 kDa c '' subunit in Figure 1) (Nish
igori H et al., Genomics. 50: 222-228, 1998), and was found to be expressed on the cell membrane of various tumors (FIG. 1). So, except for c ''
It was examined whether the c-subunit (SEQ ID NO: 5 (derived from mouse), SEQ ID NO: 8 (derived from human)) and a subunit (SEQ ID NO: 11), which are V-ATPase constituent subunits, were also expressed in tumors. Furthermore, an antibody against a peptide exposed outside the membrane of the c subunit was prepared, and the antitumor effect was examined using this antibody. Based on these findings, it was concluded that the use of antibodies against not only the c ″ subunit of V-ATPase but also the extramembrane peptides of the c subunit and the a subunit resulted in an antitumor effect. Therefore, a group of V-ATPase inhibitors that have been used for completely different purposes (all of which have a V-ATPase inhibitor action among all drugs except bafilomycin) has a new use as an antitumor agent. We examined whether there was. Furthermore, among the proton pumps other than V-ATPase, H + / K + -ATPase inhibitors that have been used for applications completely different from anticancer drugs such as gastric acid suppression (H + / K + -ATPase inhibitory action among all drugs except lansoprazole) And the antitumor effect thereof was examined. Furthermore, H + / Cl- symporter or V-ATPase
The antitumor effect of a group of drugs called uncouplers was also investigated. From these results, it has been found that any compound that inhibits the proton pump has a novel pharmacological action called an antitumor action, and has invented a promising group of drugs as an antitumor drug. In addition, the partial nucleotide sequence of a tumor-specific antigen peptide of a spontaneous mouse tumor was determined, and it was found that the amino acid at the epitope site was different from that of the tumor-specific antigen peptide, thereby completing the present invention.

That is, the present application provides the following inventions. (1) A diagnostic, prophylactic or therapeutic agent for a tumor, comprising as an active ingredient at least one selected from the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising an amino acid sequence in which at least one or several amino acids have been deleted, substituted or added in the amino acid sequence or a sequence containing at least a partial sequence thereof, and which reacts specifically with tumors

(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof; and (d) one or several amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof. Or a protein comprising an added amino acid sequence and reacting specifically with tumor (e) a protein comprising an amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) an amino acid sequence represented by SEQ ID NO: 11 or A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in a partial sequence, and which reacts specifically with tumors

Here, the above-mentioned tumor includes thyroid cancer, breast cancer, stomach cancer, esophagus cancer, oral cancer, colon cancer, rectum cancer, anal cancer,
Pancreatic cancer, lung cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nervous system tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer ,
Selected from the group consisting of gallbladder cancer, renal pelvic tumor, ureteral cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma At least one type (the same applies hereinafter).

(2) A diagnostic, prophylactic or therapeutic agent for a tumor, comprising an antibody against the protein as an active ingredient. (3) A diagnostic or therapeutic agent for a tumor, comprising at least one selected from the group consisting of the following DNAs (a) to (f) or an antisense DNA thereof. (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof (b) Hybridizing with the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof under stringent conditions, and being tumor-specific DNA that encodes a protein that reacts with

(C) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 7 or a partial sequence thereof; (d) hybridizing under stringent conditions to the nucleotide sequence represented by SEQ ID NO: 7 or a partial sequence thereof, and DNA encoding a protein that reacts specifically with tumor (e) DNA consisting of the base sequence represented by SEQ ID NO: 10 or a partial sequence thereof (f) Stringent with the base sequence represented by SEQ ID NO: 10 or a partial sequence thereof DNA encoding a protein that hybridizes under conditions and reacts specifically with tumor

(4) At least one selected from the group consisting of RNAs of the following (a) to (f) or an antisense RN thereof:
A diagnostic or therapeutic agent for a tumor, comprising A. (a) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 or a partial sequence thereof (b) Hybridizing with the nucleotide sequence represented by SEQ ID NO: 3 or a partial sequence thereof under stringent conditions, and being tumor-specific (C) RNA comprising the base sequence represented by SEQ ID NO: 9 or a partial sequence thereof

(D) RNA encoding a protein which hybridizes with the nucleotide sequence represented by SEQ ID NO: 9 or a partial sequence thereof under stringent conditions and reacts specifically with tumor (e) is represented by SEQ ID NO: 12 (F) RNA encoding a protein that hybridizes with the nucleotide sequence represented by SEQ ID NO: 12 or a partial sequence thereof under stringent conditions and reacts specifically with tumor

(5) A diagnostic or therapeutic agent for a tumor, comprising a recombinant vector containing the DNA or RNA, or a transformant containing the recombinant vector. (6) A method for detecting a tumor-specific antigen by reacting the DNA, RNA, recombinant vector or transformant with a body fluid or a tumor cell. (7) Leukocytes which react with at least one selected from the above proteins, or leukocytes which bind to an antibody against any protein selected from the above proteins. (8) A pharmaceutical composition containing the leukocyte as an active ingredient, or a prophylactic or therapeutic agent for a tumor.

(9) The following tumor-specific antigen peptide (a) or (b): (a) a tumor-specific antigen peptide consisting of an amino acid sequence represented by Phe Ser Ala Thr Glu Pro Lys; (b) a tumor containing at least the amino acid sequence represented by Phe Ser Ala Thr Glu Pro Lys, and being tumor-specifically expressed Specific antigen peptide. (10) A tumor-specific antigen peptide that contains at least the 49th to 55th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 14, and is specifically expressed in a tumor.

(11) A tumor-specific antigen peptide of the following (a) or (b): (a) a tumor-specific antigen peptide consisting of any of the amino acid sequences represented by SEQ ID NOS: 31 to 33; (b) one or several amino acids deleted in any of the amino acid sequences represented by SEQ ID NOS: 31 to 33 , A tumor-specific antigen peptide comprising a substituted or added amino acid sequence and expressed in a tumor-specific manner.

(12) A nucleic acid encoding a protein containing the peptide according to any of (9) to (11). As the nucleic acid, the following (a) or (b) DNA or (c) or (d) R
NA. (A) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 13 (b) hybridizing with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 13 under stringent conditions, and a peptide that is specifically expressed in tumor Encodes a protein containing
DNA (c) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 15 (d) Peptide that hybridizes with RNA consisting of the nucleotide sequence represented by SEQ ID NO: 15 under stringent conditions, and is specifically expressed in a tumor Encodes a protein containing
RNA

(13) A recombinant vector containing the nucleic acid of the above (12), and a transformant containing the recombinant vector. (14) An antibody against the peptide according to any of (9) to (11). Antibodies include monoclonal or polyclonal antibodies or fragments thereof. (15) the peptide of any of (9) to (11) or the (14)
White blood cells that react with the antibodies.

(16) A pharmaceutical composition comprising, as an active ingredient, the peptide of any of (9) to (11), the antibody of (14), or the leukocyte of (15). (17) Proton pump inhibitors or proton pump uncouplers (bafilomycin, lansoprazole and BE-1
Excluding 8591. ), V-ATPase inhibitor (excluding bafilomycin), or V-ATPase uncoupler containing H + / Cl-symporter (excluding BE-18591) as an active ingredient for preventing or treating tumors Agent.

Examples of the V-ATPase inhibitor include EDTA, EGTA, conkanamycin A, conkanamycin B, O-methylconkanamycin B, N-ethylmaleimide, N, N'-dicyclohexylcarbodiimide, 7-chloro-4-nitrobenz -2-oxa-1,3-diazole, suramin, verapamil, (2Z, 4E)
-5- (5,6-dichloro-2-indolyl) -2-methoxy-N- (1,2,
2,6,6-pentamethylpiperidin-4-yl) -2,4-pentadienamide, OST-766, 5- (5,6-dichloro-2-indolyl)
-2-methoxy-2,4-pentadienamide, NO ₃ , 3'-O- (4-
(Benzoyl) benzoyl adenosine 5'-triphosphate, Destraxin B, Destraxin E, Leucanisidine, Hygrolidine, Bisphosphonate tiludronate, Diethyl pyrocarbonate, Dibromoacetophenone, p-Chloromercuric benzoic acid, Quercetin, 4-Acetamide-4 '-Isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, N-bromosuccinimide and at least selected from the group consisting of benzoyltaxinine K One type.

Examples of the H + / K + -ATPase inhibitor include rabeprazole sodium, omeprazole, pantoprazole, reminoprazole, 2-dimethylamino-4,5-dihydrothiazolo [4,5: 3,4] pyridol [1 , 2-a] benzimidazole and 2-aryl-4,5-dihydro-1H-thieno [3,2-
e] In addition to benzimidazole derivatives such as benzimidazole, (±) -5-methoxy-2-[[(4-methoxy-3,5-dimethylpyrid-2-yl) methyl] sulfinyl] -1H-imidazo
(4,5-b) pyridine, 3-amino-5-methyl-2- (2-methyl-3-
Thienyl) imidazo [1,2-a] thieno [3,2-c] pyridine, 1-
Aryl-3-substituted pyrrolo [3,2-c] quinoline, 3-butyryl-8
-Methoxy-4-[(2-thiophenyl) amino] quinoline, 2-
From [(3,5-dimethyl-4-methoxypyridyl) alkyl] -benzothiazolidine, 3-butyryl-4- [R-1-methylbenzylamino] -8-ethoxy-1,7-naphthyridine and pentagalloylglucose At least one selected from the group consisting of:

Examples of V-ATPase uncouplers containing H + / Cl- symporters include prodigiosins (BEs) such as prodigiosin, metacycloprodigiosin, prodigiosin 25-C, cycloprodigiosin and cycloprodigiosin hydrochloride. -18591), and at least one selected from the group consisting of bepridil, manganese tetraphenylporphine, and thallium chloride.

(18) A functional antitumor composition containing any of the above peptides or proteins, Phe Ser Ala Thr
Functional antitumor composition containing a plant containing the amino acid sequence represented by Asp Pro Lys, or Phe Ser Ala Thr Asp Pr
o A functional antitumor composition containing aquaporin containing the amino acid sequence represented by Lys or a fragment thereof. Examples of aquaporin fragments include at least one selected from those represented by SEQ ID NOs: 46 to 52. Hereinafter, the present invention will be described in detail.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention basically relates to gene diagnosis and gene therapy for tumors utilizing DNA or RNA encoding a protein containing a tumor-specific antigen peptide having at least 7 amino acid sequences, And V-ATPase
A prophylactic or therapeutic agent for tumors with an antibody against the c or a subunit of the above. Here, the term "antibody" in the present invention means any of monoclonal antibodies and polyclonal antibodies produced by mammals including humans, for example, and fragments of these antibodies [for example, Fab fragments, F (ab) '2 fragments,
Single chain antibody (scFv)]. Further, the present invention relates to V-ATP
ase inhibitor (except bafilomycin), H + / K + -ATPase
The present invention relates to a diagnostic, preventive or therapeutic agent for tumors, including an inhibitor (excluding lansoprazole), a V-ATPase uncoupler, or an H + / Cl-symporter (excluding BE18591).

In the present invention, first, a cell line is established from human thyroid cancer, an antigenic peptide is extracted from the cell membrane components, and a polyclonal antibody or monoclonal antibody against the peptide is prepared. Then, a monoclonal antibody that specifically reacts with thyroid cancer cells is selected. Next, a part of the nucleotide sequence of DNA or RNA encoding the tumor antigen peptide recognized by the polyclonal antibody or the monoclonal antibody is determined. Thereafter, the entire nucleotide sequence of the antigen peptide is determined using the 5′-RACE method. Then, the location of the antigen peptide in the tumor cell is confirmed, and its antigenic determinant (epitope) is determined.

Further, in the present invention, a gene diagnostic agent is developed utilizing the fact that the peptide is expressed at a high rate in tumors. The cell killing effect of the polyclonal antibody or the monoclonal antibody on the tumor is confirmed, and a peptide of the epitope is chemically synthesized, and this is used as an agent for diagnosing, preventing or treating a tumor. Furthermore, DNA or R encoding the peptide
NA and each antisense nucleic acid are used as a diagnostic, preventive or therapeutic agent for tumors. Further, leukocytes which specifically react with the above peptide or leukocytes which bind to an antibody (particularly, Fc portion) against the above peptide can also be used in the present invention.

Further, V-ATPase other than the c '' subunit
We investigate whether the c subunit and a subunit, which are constituent subunits, are also expressed in tumors. Next, an antibody against the amino acid sequence of the c subunit and the a subunit that is exposed outside the tumor cell membrane is prepared, and the antitumor effect is examined using the antibody.

An antitumor effect as a novel use of a V-ATPase inhibitor (excluding bafilomycin) will be examined.
And H + / K + -AT, a proton pump other than V-ATPase
To investigate the antitumor effects of Pase (except lansoprazole) inhibitors and V-ATPase uncouplers or H + / Cl- symporters (except BE18591). Furthermore, the partial nucleotide sequence of a tumor-specific antigen peptide of a spontaneous mouse tumor was determined,
It is examined whether this differs from the tumor-specific antigen peptide in the amino acid sequence of the epitope site.

1. Preparation of antibody against tumor-specific antigen peptide (1) Preparation of antigen First, a cell membrane component to be used as an immunogen (antigen) is prepared. Preparation of cell membrane components can be obtained as follows.

As a raw material for preparing a cell membrane component, various cancer tissues or cancer cells or established cell lines can be used. When a cancer tissue or a cancer cell is used as a raw material, the cancer tissue or the cancer cell extracted from a cancer patient such as thyroid cancer is crushed to form a cell suspension. When an established cell line is used as a raw material, a cell line established in advance is cultured in a large amount and suspended in an appropriate buffer to form a cell suspension. Examples of established cell lines include thyroid cancer cells such as TPC-1, TPC-2, and TPC-3. Subsequently, these cell suspensions are subjected to an extraction treatment by a known extraction method. For example, after the cell lysate is centrifuged, the insoluble fraction is suspended in an appropriate buffer and centrifuged. The centrifugation operation is repeated several times, and the obtained precipitate is suspended in sodium deoxyxolate or the like and stirred. The resulting precipitate is centrifuged and dissolved in a small amount of a buffer (10 mM PBS or the like).
After removing the mixed albumin, the membrane component is prepared by concentration and air drying. Next, the obtained membrane component was added to an appropriate buffer (for example, 1 M KCl, 1 mM monoiodoacetic acid,
M sodium citrate) and centrifuge to prepare the antigen.

The proton pump c ″ subunit (SEQ ID NO: 2), mouse c subunit (SEQ ID NO: 5), human c subunit (SEQ ID NO: 8), and human a subunit (SEQ ID NO: 11) ) Is used as the antigen, the whole or part of the amino acid sequence shown in each SEQ ID NO can be used as the antigen. "Partial sequence" refers to at least six amino acid sequences in each amino acid sequence. However, in the present invention, the c ″ subunit can also be prepared so as to include all or part of the amino acid sequence at positions 1 to 85 in the sequence shown in SEQ ID NO: 2. For example, the entirety of the 1st to 85th amino acid sequences may be used, or 6 to 84 partial sequences may be used. Is also good. These amino acid sequences can be obtained by peptide engineering described below, or by introducing a DNA (SEQ ID NO: 1, 4, 7, or 10) encoding these amino acid sequences or a partial fragment thereof into an appropriate host by genetic engineering techniques. Can also be obtained by expression using

(2) Preparation of polyclonal antibody against the peptide of the present invention An animal is immunized with the antigen prepared as described above. The dose of the antigen per animal is as follows for mice:
When no adjuvant is used, the amount is 0.1 to 10 mg, and when no adjuvant is used, the amount is 1 to 100 μg. Examples of the adjuvant include carriers such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.

Immunization is performed by administering to a mammal (eg, rat, mouse, rabbit, human, etc.). The site of administration is intravenous, subcutaneous or intraperitoneal. Also,
The interval of immunization is not particularly limited, and is several days to several weeks, preferably two to three weeks, and is 1 to 10 times, preferably 2 to 10 times.
Immunize three times. Then, the antibody titer was measured 6 to 60 days after the last immunization day, and blood was collected on the day showing the highest antibody titer,
Obtain antiserum. Measurement of antibody titer is performed by enzyme immunoassay (ELIS
A; enzyme-linked immunosorbent assay), radioimmunoassay (RIA; radioimmuno assay) and the like. If antibody purification from antiserum is required, ammonium sulfate precipitation, ion exchange chromatography, gel filtration,
Purification can be performed by appropriately selecting a known method such as affinity chromatography, or by combining these methods.

(3) Preparation of monoclonal antibody against the peptide of the present invention (3-1) Immunization and collection of antibody-producing cells Animals are immunized with the antigen peptide prepared as described above. If necessary, to immunize effectively,
Adjuvants (commercially available complete Freund's adjuvant, incomplete Freund's adjuvant, etc.) may be mixed as described above. Immunization is performed by administering to mammals (for example, rats, mice, rabbits, etc.). The single dose of antigen is 1 mg per mouse in mice. The administration site is mainly intravenous, subcutaneous, or intraperitoneal. Also,
The interval between immunizations is not particularly limited, and the immunization is performed at least 2-3 times at intervals of several days to several weeks, preferably at intervals of two to three weeks. And
After the final immunization, the antibody-producing cells are collected. Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, and the like, with spleen cells being preferred.

(3-2) Cell fusion In order to obtain a hybridoma, for example, cell fusion between a human or other mammalian antibody-producing cell and a myeloma cell is performed. As the myeloma cells to be fused with the antibody-producing cells, generally available cell lines derived from animals such as humans and mice can be used. As a cell line to be used, it has drug selectivity and HAT selection medium when unfused
(Including hypoxanthine, aminopterin and thymidine)
It is preferred that they have the property of not being able to survive and survive only in the state of being fused with the antibody-producing cells. For example, specific examples of myeloma cells include P3X63-Ag.8.U1 (P3U1), P3 /
Mouse myeloma cell lines such as NSI / 1-Ag4-1 and Sp2 / 0-Ag14.

Next, the myeloma cells are fused with the antibody-producing cells. Cell fusion uses serum-free DME
M, in a medium for animal cell culture such as RPMI-1640 medium, antibody-producing cells and myeloma cells at a predetermined ratio (for example, 5:
The mixture is mixed in 1), and the fusion reaction is performed in the presence of a cell fusion promoter such as polyethylene glycol or by electric pulse treatment (for example, electroporation).

(3-3) Selection and Cloning of Hybridomas A desired hybridoma is selected from the cells after the cell fusion treatment. For example, cells that grow by culturing using a medium containing hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) can be obtained as hybridomas.

Next, it is screened whether the target antibody is present in the culture supernatant of the grown hybridoma. Hybridoma screening may be performed according to a conventional method, and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected and subjected to enzyme immunoassay (ELISA; enzym
e-linked immunosorbent assay), RIA (radioimmuno a
ssay) and the like. Cloning of the fused cells is performed by a limiting dilution method or the like, and finally a hybridoma, which is a monoclonal antibody-producing cell, is established.

(3-4) Collection of Monoclonal Antibody As a method for collecting a monoclonal antibody from the established hybridoma, a usual cell culture method or the like can be adopted. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium or MEM medium containing 10% fetal bovine serum under ordinary culture conditions (for example, 37 ° C., 5% CO ₂ concentration) for 3 to 10 days. Then, an antibody is obtained from the culture supernatant.

When antibody purification is required in the above antibody collection method, known methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel chromatography are appropriately selected. Alternatively, purification can be performed by combining these methods.

Hybridoma KTC-3 (named hybridoma KTC-3), which produces a mouse monoclonal antibody (produced using thyroid cancer cells as an antigen), is available from the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Ibaraki Prefecture). 1-3 1-3 Higashi, Tsukuba City
No. FERM BP-6899, and deposited internationally under the Budapest Treaty (Original deposit date: September 30, 1998)
Day).

(3-5) Properties of the monoclonal antibody of the present invention (i) According to the result of examining cancer specificity by ELISA,
It specifically reacts with cells derived from various cancers (for example, thyroid cancer, stomach cancer, colon cancer, and kidney cancer), but does not react with normal cells. (ii) As a result of immunoelectron microscopic observation using colloidal gold particles, it reacts with an epitope present on the cancer cell membrane. (iii) As a result of the proteolytic enzyme and glycolytic enzyme treatment, the enzyme is degraded by the proteolytic enzyme, but is not affected by the sugar treatment. (iv) As a result of examining the cell killing effect on cancer cells by treating the monoclonal antibody with papain, even a Fab fragment has a cell killing effect on cancer cells. Therefore, each Ig class incorporating the Fab, for example, IgM (μ chain), IgG (γ chain),
Any of IgA (α chain), IgE (ε chain) and IgD (δ chain), or a carrier molecule bound to Fab is also effective. (v) From the results of the above (i) to (iv) and the results of analysis using the synthetic peptide, at least the amino acid sequence: Phe Ser Ala Th
r Reacts with peptides having Asp Pro Lys.

2. Analysis of gene encoding tumor antigen protein (1) c '' subunit of V-ATPase (1-1) Preparation and screening of cDNA library encoding tumor antigen protein The tumor-specific antigen peptide of the present invention has an amino acid sequence : Phe
It consists of Ser Ala Thr Asp ProLys, and has the 118th amino acid sequence (SEQ ID NO: 2) of the tumor antigen protein.
It corresponds to the ~ 124th amino acid (referred to as "M1"). Such amino acid sequences include, for example, those containing amino acids 117 to 125 of the amino acid sequence (`` A
la9 "), containing amino acids 116-125
(Referred to as "Ala10") and those containing amino acids 115 to 134 (referred to as "Gly20").

The amino acid sequences of these peptides are shown below. M1: Phe Ser Ala Thr Asp Pro Lys (SEQ ID NO: 16) Ala9: Pro Phe Ser Ala Thr Asp Pro Lys Ala (SEQ ID NO: 17) Ala10: Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala (SEQ ID NO: 18) Gly20: Ala Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala Ile Gly His Arg Asn Tyr His Ala Gly (SEQ ID NO: 19)

In the present invention, a protein partially containing these peptides (also referred to as “tumor antigen protein”)
In order to obtain DNA encoding the protein, RNA is prepared from tumor cells, and the DNA is cloned. Preparation of the tumor antigen protein mRNA can be performed by a commonly used technique. For example, thyroid cancer cells and the like are treated with a guanidine reagent, a phenol reagent, etc. to obtain total RNA, and then an affinity column method using oligo dT-cellulose or poly U-sepharose using Sepharose 2B as a carrier, or a batch method. To obtain poly (A +) RNA (mRNA). Using the resulting mRNA as a template, a single-stranded cDNA is synthesized using an oligo dT primer and a reverse transcriptase, and then a double-stranded cDNA is synthesized from the single-stranded cDNA. By incorporating the thus obtained double-stranded cDNA into an appropriate expression vector (for example, λgt11) to produce a recombinant vector, cD
An NA library can be obtained.

The screening method for selecting a strain having the desired DNA from the cDNA library obtained as described above includes, for example, a method using a monoclonal antibody. On the other hand, RNA encoding a tumor antigen protein can be obtained by incorporating a target DNA into a plasmid vector, purifying the plasmid, and then using an in vitro transcription system. Further, a partial sequence of the above DNA or RNA can be used in the present invention. The partial sequence means a sequence corresponding to a partial sequence of the amino acid or a complementary sequence thereof.

(1-2) Preparation of Mutant Gene Encoding Tumor Antigen Protein In the present invention, at least a part (excluding the 118th to 124th amino acids) of the amino acid sequence (SEQ ID NO: 2) of the tumor antigen protein is mutated. Can also be introduced. Therefore, the mutant is also included in the protein of the present invention as long as it exhibits tumor-specific antigenicity. In order to introduce a mutation into an amino acid, a technique for introducing a mutation into the nucleotide sequence of a gene encoding the amino acid is employed.

A mutation can be introduced into a gene by a known method such as the Kunkel method or the Gapped duplex method. For example, a mutation is introduced based on a site-directed mutagenesis method using a mutant oligonucleotide as a primer, and a mutation introduction kit (for example, Mutant-K (TAKARA
Mutant-G (TAKARA), TAKARA LA PCR in
Mutations can also be introduced using an in vitro Mutagenesis series kit).

(1-3) Determination of Nucleotide Sequence The nucleotide sequence of the gene obtained as described in (1-1) or (1-2) is determined. The nucleotide sequence can be determined by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method using DNA polymerase. Although the nucleotide sequence of the S1 antigen was determined by the above method, since the nucleotide sequence of the 5 ′ region of the cDNA has not been determined, the entire nucleotide sequence is determined using 5′-RACE described below.

(1-3) Determination of Nucleotide Sequence The nucleotide sequence of the gene obtained as described in (1-1) or (1-2) is determined. The nucleotide sequence can be determined by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method using DNA polymerase. Although the nucleotide sequence of the S1 antigen was determined by the above method, since the nucleotide sequence of the 5 ′ region of the cDNA has not been determined, the entire nucleotide sequence is determined using 5′-RACE described below.

(1-4) Isolation of 5 ′ region of cDNA and determination of total nucleotide sequence After extracting total RNA from TPC-1 cells by GTC / Li method,
Poly (A) + RNA was purified by o (dT) column chromatography. Next, using 0.5 μg of the poly (A) + RNA as a template
5 'of S-1 tumor antigen cDNA using BRL's 5'-RACE system
The region was amplified by PCR and isolated. That is, the gene-specific oligo DNA (GSP1) designed in the region where the sequence has already been determined (sequence: 5′-CCC CAC CCA CAC ATA ACT T
C-3 ′ (SEQ ID NO: 34)) was used as a primer to synthesize a single-stranded cDNA using reverse transcriptase. Next, Td is added to the 3 'end of the cDNA.
A dC polymer was added with T (terminal deoxynucleotidyl transferase) and dCTP. Using it as a template, an adapter primer with dG polymer (sequence: 5'-CUA CUA CUA CUA GGC CAC GCG TCG ACTAGT
ACG GGI IGG GII GGG IIG-3 '(SEQ ID NO: 36)) and GSP1
Designed upstream of GSP2 (sequence: 5'-TAG ACA GGC CTA CGG
The first PCR was performed using TGA-3 ′ (SEQ ID NO: 35)) as a primer. The sequence of SEQ ID NO: 36 is DN
A binding molecule between A and RNA, and I represents inosine. In addition, an adapter primer and a GS designed further upstream
P3 (sequence: 5'-GGA GTA GCC TGC ATG-3 '(SEQ ID NO: 3
7)) and a second PCR was performed. The gene fragment obtained as described above was subcloned into the plasmid Bluescript II, and the nucleotide sequence was determined by the dideoxy method.

SEQ ID NO: 1 exemplifies the nucleotide sequence of the DNA encoding the tumor antigen protein of the present invention, SEQ ID NO: 2 exemplifies the amino acid sequence of the protein, and SEQ ID NO: 3 shows the nucleotide sequence of the RNA encoding the tumor antigen protein of the present invention. The base sequence is exemplified. However, the 11th protein of this amino acid sequence
One or several amino acids in the amino acid sequence may be mutated such as deletion, substitution, addition, etc., as long as the peptide having the sequence of positions 8 to 124 is specifically expressed in cancer cells.

For example, at least one, preferably about 1 to 10, and more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be deleted. At least one in the sequence,
Preferably about 1 to 10, more preferably 1 to 5 amino acids may be added, or at least one, preferably one, of the amino acid sequence represented by SEQ ID NO: 2.
About to 10, more preferably 1 to 5 amino acids may be substituted with another amino acid.

Furthermore, DNAs or RNAs encoding a protein containing a peptide that hybridizes with the above DNA (SEQ ID NO: 1) or RNA (SEQ ID NO: 3) under stringent conditions and is specifically expressed in a tumor are also included in the present invention. DNA or RNA. Stringent conditions mean that the sodium concentration
0.1 × SSC, which means that the temperature is 50 ° C. Once the nucleotide sequence of the DNA or RNA of the present invention is determined, thereafter, the DNA or RNA of the present invention is obtained by chemical synthesis or by PCR using a primer synthesized from the determined nucleotide sequence.
RNA can be obtained.

The DNA of the present invention (337 to 9 of SEQ ID NO: 1)
The 86th sequence) was introduced into E. coli K12 (name: pRIT2
T-625), and has been deposited internationally under the Budapest Treaty with the Institute of Biotechnology and Industrial Technology (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture) under the Budapest Treaty (Original deposit date: 1998) (1998) September 30).

Further, the DNA of the present invention (the entire sequence of SEQ ID NO: 1) was introduced into Escherichia coli (name: SSY), and the Institute of Biotechnology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) was used.
No.), deposited as FERM P-17801 (Deposit date:
March 30, 2000). (2) c-subunit of V-ATPase DNA encoding the c-subunit of V-ATPase can be obtained as follows. That is, RT-PCR, TOYO
This was performed using the RT-PCR high kit from BO. That is, T
PCR was performed using cDNA obtained by reverse transcription of 25 ng of total RNA extracted from PC-1 cells as a template. PCR is
Using the Fc primer and Rc primer shown below, 94 ° C 30
This was performed 33 times with the conditions of second, 56 ° C. for 2 minutes and 72 ° C. for 1 minute as one cycle. The gene fragment obtained as described above was subcloned into the plasmid Bluescript II, and the nucleotide sequence was determined by the dideoxy method. Forward primer

Fc primer (forward primer c): 5'-GG
CCGCCATGGTCTTCA-3 '(SEQ ID NO: 38) (corresponding to the 288-304 sequence of the nucleotide sequence described in SEQ ID NO: 7) Reverse primer Rc primer (reverse primer c): 5'-TGAGGAGGGGTGGT
CTTTAC-3 ′ (SEQ ID NO: 39) (SEQ ID NO: 7
(A sequence complementary to the 31st to 750th sequence)

SEQ ID NO: 7 shows the nucleotide sequence of the DNA encoding the human c subunit, SEQ ID NO: 8 shows the amino acid sequence of the subunit, and SEQ ID NO: 9 shows the nucleotide sequence of the RNA encoding the c subunit. For example. In addition, SEQ ID NO: 4 illustrates the nucleotide sequence of the DNA encoding the mouse c-subunit, SEQ ID NO: 5 illustrates the amino acid sequence of the subunit, and SEQ ID NO: 6 illustrates the RNA encoding the c-subunit.
Is exemplified. However, mutation such as deletion, substitution, addition, etc. may occur in one or several amino acids in the amino acid sequence as long as the protein comprising these amino acid sequences is expressed specifically in cancer cells. The mode of mutation is the same as described above.

(3) V-ATPase a Subunit DNA encoding the V-ATPase a subunit can be obtained as follows. That is, RT-PCR, TOYO
This was performed using the RT-PCR high kit from BO. That is, T
PCR was performed using cDNA obtained by reverse transcription of 25 ng of total RNA extracted from PC-1 cells as a template. PCR is
Using the Fa primer and Ra primer shown below,
This was performed 33 times with the conditions of second, 56 ° C. for 2 minutes and 72 ° C. for 1 minute as one cycle. The gene fragment obtained as described above was subcloned into the plasmid Bluescript II, and the nucleotide sequence was determined by the dideoxy method. Forward primer

Fa primer (forward primer a): 5'-CC
ACCATGGGGGAGCT-3 '(SEQ ID NO: 40) (corresponding to the sequence from position 164 to 179 of the nucleotide sequence described in SEQ ID NO: 10) Reverse primer Ra primer (reverse primer a): 5'-CAAGTCTGGATCCG
CCATC-3 ′ (SEQ ID NO: 41) (5th nucleotide sequence of SEQ ID NO: 10)
(A sequence complementary to the 91st to 609th sequences)

D encoding the a subunit in SEQ ID NO: 10
For the nucleotide sequence of NA, SEQ ID NO: 11 exemplifies the amino acid sequence of the subunit, and SEQ ID NO: 12 exemplifies the nucleotide sequence of RNA encoding the a subunit. However, as long as the protein consisting of this amino acid sequence is specifically expressed in cancer cells,
Mutations such as deletion, substitution, and addition may occur in one or several amino acids in the amino acid sequence. The mode of mutation is the same as described above.

(4) Erythroleukemia cell EL-4 In the present invention, mouse erythroleukemia cell EL-4 can be exemplified as an antigen that can be derived from a cancer cell. The DNA encoding the protein derived from EL-4 is RT-
It can be obtained by PCR. That is, c obtained by reverse transcription of total RNA extracted from mouse EL-4 cells
A PCR reaction is performed using DNA as a template, and the obtained gene fragment is subcloned into an appropriate plasmid, and the nucleotide sequence is determined. The method for inserting the gene fragment into the plasmid, the method for determining the nucleotide sequence, and the like are the same as described above.

Thereafter, the DNA base sequence of the gene was analyzed,
The sequence corresponding to the tumor antigen epitope is specified. The sequence corresponding to the tumor-specific antigen of EL-4 cells is Phe Ser Ala Thr
Glu Pro Lys (SEQ ID NO: 30), and the fifth Asp in the amino acid sequence of SEQ ID NO: 16 is Gl
is replaced by u.

SEQ ID NO: 13 exemplifies the nucleotide sequence of the DNA encoding the tumor antigen protein of EL-4, SEQ ID NO: 14 exemplifies the amino acid sequence of the protein, and SEQ ID NO: 15 the nucleotide sequence of the RNA encoding the protein Is exemplified. However, mutations such as deletion, substitution, and addition may occur in one or several amino acids in the amino acid sequence as long as the protein comprising the amino acid sequence is expressed specifically in cancer cells.
The mode of mutation is the same as described above.

The DN encoding the EL-4 antigen protein
A (SEQ ID NO: 13) was introduced into E. coli (name: EL-
4) Deposited with FERM P-17800 at the Research Institute of Biotechnology and Industrial Technology (1-3-3 Higashi, Tsukuba, Ibaraki Prefecture) (Deposit date: March 30, 2000) ).

3. Preparation of Recombinant Vector The recombinant vector used in the present invention can be obtained by ligating (inserting) a gene (DNA or RNA) encoding the tumor antigen protein of the present invention into an appropriate vector. A vector for inserting the gene of the present invention,
There is no particular limitation as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA.

Plasmid DNAs include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119,
Bluescript II SK +/-, pGEM4, pSP64, pSP65, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.). Phage (λgt10,
λgt11, M13mp18, M13mp19, etc.). Also,
Animal viruses such as retrovirus, adenovirus or vaccinia virus, or insect virus vectors such as baculovirus can also be used. Furthermore, a fusion plasmid in which GST, GFP, His-tag, Myc-tag, etc. are linked can also be used. In order to insert the gene of the present invention into a vector, first, a method in which purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA at a restriction enzyme site or a multiple cloning site, and ligated to a vector. Adopted.

The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, the vector of the present invention may be ligated to a vector containing a promoter, a gene of the present invention, and optionally a cis element such as an enhancer, a splicing signal, a polyA addition signal, a selection marker, a ribosome binding sequence, and the like. it can. In addition, examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

4. Preparation of Transformant The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention.
For example, the genus Escherichia, such as Escherichia coli, Bacillus subtilis (Bacillus)
subtilis), Pseudomonas putida (Ps
eudomonas putida), bacteria belonging to the genus Pseudomonas, Saccharomyces cerevisiae (Saccharomyc
es cerevisiae), Schizosaccharomyces pombe (Schizos
accharomyces pombe), COS cells, CH
Examples include animal cells such as O cells, and insect cells such as Sf9 and Sf21.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and comprises a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Further, a gene controlling a promoter may be included. As Escherichia coli, for example, Escherichia
E. coli (Escherichia coli) K12, DH1, etc., and examples of Bacillus subtilis include Bacillus subtilis (Bacillus su
btilis) MI 114, 207-21 and the like.

Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example trp
Promoter, lac promoter, PL promoter, PR
A promoter such as a promoter derived from Escherichia coli or a phage is used. An artificially designed and modified promoter such as a tac promoter may be used.

The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions (Cohen,
SNet al .: Proc. Natl. Acad. Sci., USA, 69: 2110-
2114 (1972)), electroporation method (Becker, D.
M. et al .: Methods. Enzymol., 194: 182-187 (199
0)) and the like.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe
Are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast, for example,
gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5
Promoter, PGK promoter, GAP promoter, AD
H promoter, AOX1 promoter and the like.

The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, an electroporation method, a spheroplast method (Hin
nen, A. et al .: Proc. Natl. Acad. Sci., USA, 75: 1.
929-1933 (1978)), lithium acetate method (Itoh, H .: J. Bact
eriol., 153: 163-168 (1983)). When animal cells are used as hosts, COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse myeloma cells, and the like are used. SRα promoter as promoter,
An SV40 promoter, an LTR promoter or the like may be used, and an early gene promoter of human cytomegalovirus may be used.

Examples of a method for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method. When an insect cell is used as a host, Sf9 cells, Sf21 cells and the like can be mentioned. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method,
An electroporation method or the like is used.

5. Production of Tumor Antigen Protein A protein containing the peptide of the present invention (tumor antigen protein) is obtained by transforming the transformant prepared in 4 above in vivo.
Alternatively, it can be produced by culturing in vitro and collecting from the culture. The term “culture” means any of a culture supernatant, a cultured cell, a cultured cell, and a crushed cell or cell. The method for culturing the transformant of the present invention is performed according to a usual method used for culturing a host.

A culture medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the microorganism. If the medium can be performed efficiently,
Either a natural medium or a synthetic medium may be used.

Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. As a nitrogen source, ammonia,
In addition to ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like can be mentioned. Examples of the inorganic substance include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

The cultivation is usually carried out at 37 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture, the pH is maintained at 7-7.5. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution or the like. During culture, antibiotics such as ampicillin and tetracycline may be added to the medium as needed.

When a microorganism transformed with an expression vector using an inducible promoter is cultured, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using a Lac promoter, culturing a microorganism transformed with an expression vector using a trp promoter, such as isopropyl-β-D-thiogalactopyranoside (IPTG). At times, indole acrylic acid (IAA) or the like may be added to the medium.

As a medium for culturing a transformant obtained using animal cells as a host, generally used RPMI-1
640 medium, DMEM medium or a medium obtained by adding fetal bovine serum or the like to these mediums. Culture is usually performed with 5% CO ₂
Perform in the presence at 37 ° C for 1-30 days. During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.

After the culture, when the tumor antigen protein is produced in the cells or cells, the cells are extracted by disrupting the cells or cells. When the tumor antigen protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using a general biochemical method used for protein isolation and purification, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, or the like, alone or in appropriate combination, in the culture. , A tumor antigen protein can be isolated and purified.

6. Peptide Chemical Synthesis Peptides or proteins (such as peptides) used in the present invention can be obtained by conventional peptide chemical synthesis. In addition to the amino acid sequence of the peptide or the like used in the present invention, even if one or several (preferred numbers are the same as described above) mutation, such as deletion, substitution or addition, in the amino acid sequence, As long as the mutated peptide or the like is cancer cell-specific, it is included in the scope of the present invention.

The above-mentioned peptides and the like also include salts thereof. When chemically synthesizing a peptide or the like used in the present invention, the peptide can be synthesized by a conventional method of synthesizing a peptide. For example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method,
Carboimidazole method, oxidation-reduction method and the like can be mentioned. Further, for the synthesis, any of a solid phase synthesis method and a liquid phase synthesis method can be applied.

That is, amino acids which can constitute a peptide or the like are condensed with each other, and when the product has a protecting group, the protecting group is eliminated to synthesize the desired peptide or the like. Any known method may be used for the condensation method and the removal of the protecting group [for example, Bodanszky, M and MA O
ndetti, Peptide Synthesis, Interscience Publisher
s, New York (1966), Schroeder and Luebke, The Pept
ide, Academic Press, New York (1965), Nobuo Izumiya and others,
See Basics and Experiments of Peptide Synthesis, Maruzen (1975), etc.]. After the reaction, the target peptide or the like can be purified by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.

The salts of peptides and the like are preferably physiologically acceptable acid addition salts or basic salts. Examples of the acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, and apple. Salts with organic acids such as acids, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid and the like can be mentioned. Examples of the basic salt include sodium hydroxide, potassium hydroxide,
Examples thereof include salts with inorganic bases such as ammonium hydroxide and magnesium hydroxide, and salts with organic bases such as caffeine, piperidine, trimethylamine and pyridine.

Salts can be prepared using a suitable acid such as hydrochloric acid, or a suitable base such as sodium hydroxide. For example, they can be prepared by treatment in water or in a liquid containing an inert water-miscible organic solvent such as methanol, ethanol or dioxane using standard protocols. The processing temperature is 0 to 10
0 ° C., preferably room temperature.

The above-mentioned peptides and the like usually have a carboxyl (—COOH) group or carboxylate (—COO ⁻ ) at the C-terminus, but have an amide (—CONH ₂ ) or ester (—COOR) at the C-terminus. Is also good. Here, as R in the ester,
Examples thereof include an alkyl group having 1 to 12 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, an aryl group having 6 to 12 carbon atoms, and an aralkyl group having 7 to 12 carbon atoms.

Further, the peptides and the like also include those in which the amino group of the N-terminal alanine residue is protected with a protecting group, and complex peptides such as glycopeptides having a sugar chain bonded thereto. The biochemical and physicochemical properties of peptides and the like can be analyzed by mass spectrometry, nuclear magnetic resonance, electrophoresis, high performance liquid chromatography, and the like.

7. Tumor-specific antigen peptide or protein-reactive leukocyte or antibody-reactive leukocyte In the present invention, `` leukocyte '' refers to a blood cell component collected from a host vaccinated with the tumor-specific antigen peptide or protein or a cancer-bearing host. Means lymphocytes (T lymphocytes, B lymphocytes), dendritic cells, neutrophils,
It is composed of eosinophils, basophils and monocytes. The leukocyte reacts with a peptide or protein of each subunit of V-ATPase or an EL-4 cell (referred to as a peptide-reactive leukocyte), and binds to an antibody (preferably the Fc portion) against the peptide or the like. (Referred to as antibody-reactive leukocytes or antibody-binding leukocytes). further,
Those belonging to these subgroups of leukocytes are also included in the leukocytes of the present invention. In the present invention, among these cell components, lymphocytes, monocytes, granulocytes and dendritic cells are preferred.

The term “peptide-reactive leukocyte” means a cell that presents all amino acids or a part of the antigenic expression part (epitope) of the peptide to the major histocompatibility complex (MHC) on the cell surface, By “antibody-reactive leukocyte” is meant a leukocyte capable of binding to an antibody via a receptor for the antibody (present on the surface of the leukocyte). The leukocyte of the present invention can be obtained by the following treatment.

In the case of lymphocytes, humans or animals (mouse, rat, guinea pig, hamster, rabbit, cat,
The blood, spleen, lymph nodes, etc. of experimental animals such as dogs, pigs, monkeys, etc.) can be used as the collection source. It is prepared from peripheral blood lymphocytes (PBL), lymph node cells, tumor infiltrating lymphocytes (TIL), tumor local lymph node cells, etc. contained in these tissues or organs. That is, blood collected from a human or animal, or spleen or lymph node collected by laparotomy of an animal is suspended in an appropriate cell culture solution. As the cell culture medium, a basic medium generally used for culturing animal cells, for example, a minimum basic medium (MEM), an RPMI-1640 medium, a Ham F12 medium, a Dulbecco's modified Eagle medium (DMEM)
And the like. Cell culture medium contains fetal calf serum (FC
It is preferable to contain S) and the like.

When blood is used as a source, a usual lymphocyte separation method, for example, Ficoll density gradient centrifugation, Perc
A lymphocyte fraction is obtained by oll density gradient centrifugation or the like.
When the spleen, lymph node, etc. are used as the collection source, cut these tissues or organs with scissors, leave the spleen as it is, and treat various organs and tissues with enzymes such as collagenase or DNase. Alternatively, after treatment with a chelating agent such as EDTA, each is placed on a metal mesh and gently pressed from above to allow the internal lymphocytes to migrate out of the tissue. The cells that have passed through the metal mesh are gently pipetted, left to stand for a while, and then the supernatant is obtained. Free lymphocytes are obtained by removing the tissue fragments and cell aggregates in the sediment.

In the case of dendritic cells, the above-described lymphocyte suspension is cultured in a Petri dish for 1 hour to obtain a cell group adhered to the Petri dish wall. After removing the non-adherent cells, the adherent cells are collected by adding a chelating agent. The cell group thus obtained contains dendritic cells, monocytes (macrophages) and neutrophils at a ratio of about 10%, about 40%, and about 40%, respectively. Monocytes and neutrophils are phagocytosed by coexistence of magnetic particles in these three types of cells. Magnetic particle phagocytic cells are collected in a magnetic field and removed to obtain dendritic cells.

In the case of monocytes and neutrophils, monocytes are obtained from the above phagocytic cell group using an anti-MHC class II antibody or an anti-CD11b antibody. Neutrophils are obtained by the action of an anti-CD157 antibody. In the case of eosinophils or basophils, the above-mentioned non-adherent cell group has anti-CD116 antibody, and basophils are anti-CDW125 antibody or anti-CDW antibody.
Obtained after reacting 128 antibodies. The subgroup of leukocytes includes granulocytes, lymphocytes and monocytes. These subgroups are distinguished morphologically to obtain content. In addition, in order to separate each group, it is prepared by utilizing the difference in wall adhesion ability and surface layer structure.

[0102] Of the leukocytes obtained as described above,
Confirmation of having the function of binding to the peptide or the like,
It is performed by confirming the action of enhancing nucleic acid synthesis ability (antigenesis phenomenon) after antigen binding, and confirming that it has the function of binding to the antibody of the present invention (especially the Fc portion), (Opsonizing effect) and the like.

8. Antisense oligomer used in the present invention,
It is obtained by designing a nucleic acid compound based on the base sequence of a gene serving as a template, and evaluating the effect of the compound based on the expression level of a gene encoding a tumor-specific antigen peptide.

That is, a compound having a sequence complementary to the partial sequence of the gene encoding the tumor-specific antigen peptide determined as described above is prepared by a synthetic chemical technique or the like. Then, to evaluate whether the compound effectively inhibits the expression of a tumor-specific antigen gene,
Perform screening. A nucleic acid compound containing a nucleotide sequence that inhibits the expression of a tumor-specific antigen gene is used as a diagnostic, therapeutic or prophylactic agent for tumors. Examples of the nucleic acid compound include a natural type, a phosphorothioate type, a phosphorodithioate type, a methylphosphonate type, a phosphoramidate type, an H-phosphonate type, a triester type, an α-anomeric type, and a peptide nucleic acid.

The screening of a nucleic acid compound can be performed by adding the nucleic acid compound synthesized as described above to a transcription / translation system in which a tumor-specific antigen peptide gene is present, and examining the inhibition of gene expression. Alternatively, a nucleic acid compound can be added to a translation system in which RNA encoding the tumor-specific antigen peptide is present, and the inhibition of the expression of the tumor-specific antigen peptide can be examined.

A method for confirming the production of a tumor-specific antigen peptide by the above-mentioned transcription / translation system includes an enzyme immunoassay using an antibody against the peptide. For example, after immobilizing an antibody on a microtiter plate, a tumor-specific antigen peptide gene is added and reacted. Then, an antibody labeled with horseradish peroxidase is added, the mixture is allowed to stand at room temperature, a substrate (such as diaminobenzidine) is added, color is developed, the absorbance is measured, and the content of the tumor-specific antigen peptide is evaluated.

In order to inhibit the expression of a gene, it is necessary that the nucleic acid compound has a nucleotide sequence complementary to at least 10 bases in the gene, and 15 to 20 nucleotides are preferable. . The resulting antisense oligomer is used for the antitumor treatment described below.

9. Pharmaceutical composition, diagnostic, prophylactic or therapeutic agent for tumor (1) Peptide or protein or antibody Peptide or protein (referred to as peptide, etc.)
Alternatively, the pharmaceutical composition containing an antibody as an active ingredient and the diagnostic, prophylactic or therapeutic agent for tumors may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and other surfactants acceptable as pharmaceutical additives And artificial cell structures such as liposomes. The additives to be used are appropriately or in combination selected from the above according to the dosage form of the present invention.

When the peptide or the like or the antibody of the present invention is used as a diagnostic, prophylactic or therapeutic agent for tumors, the subject to be used is not particularly limited. For example, cancer, sarcoma,
Diagnosis, treatment or prevention of at least one kind of tumor such as a benign tumor can be used for a specific purpose. These diseases may be used alone, in combination, or in combination with other diseases other than those described above, to which the peptide of the present invention can be used.

Here, when the peptide or the like or the antibody of the present invention is used for the diagnosis, prevention or treatment of cancer, these cancer types are not particularly limited. For example, thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cavity Cancer, colorectal cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer, renal cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central or peripheral nerve tumor (meningioma, glioma, Brain, spinal cord, including acoustic nerve tumors and neurofibrosarcomas
Peripheral nerve tumors), gingival cancer, pharyngeal cancer, jaw cancer (including maxillary cancer; squamous cell carcinoma), mediastinal tumor (including thymic cancer), liver cancer,
Bile duct cancer, gallbladder cancer, renal pelvis tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma (uterine cancer) At least one selected from the group consisting of sarcomas). However, not only a single cancer but also a combination of multiple cancers is included.

The diagnostic, prophylactic or therapeutic agent containing the peptide or the like or the antibody of the present invention can be administered orally or parenterally for the peptide or the like and parenterally for the antibody. . When the peptide or the like of the present invention is orally administered, a solid preparation such as tablets, granules, powders, and pills, or a liquid preparation such as a liquid or a syrup may be used. In particular, granules and powders can be in unit dosage form as capsules, or in the case of liquid preparations as dry products to be re-dissolved when used.

Of these dosage forms, oral solid preparations usually contain additives such as binders, excipients, lubricants, disintegrants, wetting agents and the like generally used in pharmaceutical preparations in their compositions. .
In addition, oral liquid preparations are usually used in those compositions in the form of stabilizers, buffers, flavoring agents, preservatives,
Contains additives such as fragrances and coloring agents.

When the peptide or the like of the present invention is administered parenterally, an injection, suppository or the like may be used. For injections,
It may be provided in the form of a unit dose ampoule or a multi-dose container, and may be a powder reconstituted with a suitable carrier for use, for example, sterile pyrogen-free water. These dosage forms usually contain additives, such as emulsifiers and suspending agents, which are generally used in pharmaceutical compositions. Injection techniques include, for example, intravenous infusion, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intradermal injection, and intratumoral injection. The dose varies depending on the age of the subject, the route of administration, and the number of administrations, and can be varied over a wide range.

In this case, the effective amount administered as a combination of the effective amount of the peptide of the present invention and a suitable diluent and a pharmacologically usable carrier is 4 times per 1 kg of body weight at a time.
μg to 20 μg, administered at 1 to 4 week intervals.
In addition, the effective amount of antibody is 100 μg
100100 mg, given at 1 to 4 week intervals.

(2) Gene Therapy In the present invention, each subunit of ATPase (c ", c or a
A gene (including DNA, RNA or a partial sequence or an antisense nucleic acid thereof) encoding a subunit) or an EL-4 tumor-specific antigen protein (hereinafter also referred to as a tumor-specific antigen gene) is used as a gene therapy agent be able to. In this case, in addition to a method of directly administering the gene of the present invention by injection, a method of administering a vector or plasmid into which the gene has been incorporated can be mentioned.

(2-1) Gene Therapy Using Vectors The vectors include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors,
Vaccinia virus vectors, retrovirus vectors and the like can be mentioned, and by using these virus vectors, administration can be performed efficiently. In addition, the tumor-specific antigen gene is introduced into phospholipid vesicles such as liposomes,
A method of administering the liposome can also be adopted. Since liposomes are closed vesicles containing biologically degradable materials, the genes are retained in the aqueous layer or lipid bilayer inside the liposomes by mixing the liposomes with the genes (liposome-gene complex ). Next, when the complex is cultured with cells, the genes in the complex are incorporated into the cells (lipofection method). And
The obtained cells are administered by the following administration method.

[0117] The dosage form of the gene therapy agent of the present invention can be not only general systemic administration such as intravenous or intraarterial administration, but also local administration to each tissue where a tumor exists. In addition, dosage forms in combination with catheter techniques, surgery and the like can be employed. The dose of the gene therapy agent of the present invention varies depending on age, sex, symptoms, administration route, number of administrations, dosage form, but usually, for example, the dose in the case of adenovirus is 10 ¹⁰ to 10 per day. It is about ¹¹ PFU and is administered every 1 to 4 weeks.

(2-2) Tumor therapeutic agent using plasmid DNA When plasmid DNA is directly administered intravenously, the DNA
It is difficult to express the gene because it is immediately degraded by DNase or the like. So, plasmid
When DNA is used, it is preferable to employ a direct injection method of plasmid DNA. Compared to the method using a viral vector, this method is: 1.Since the purification of the vector is simple, it can be prepared in a large amount in a short time. Unlimited, 3. DNA is a highly stable molecule, so it is easy to handle and can be stably expressed even after reaching the target tumor tissue. 4. Vector can be integrated into the host genome Sex is extremely rare and there is little risk of mutation of the host's gene during integration.
5.Because DNA itself has no toxicity or immunogenicity, there is no danger of an inflammatory response caused by the vector, and since there is little chance of an immune response to the vector, repeated administration is possible. Have (Ve
rma IM et al., Nature 389: 239-242, 1997).

The direct DNA injection method is very simple.
That is, the purified plasmid DNA is dissolved in physiological saline or the like, and is directly injected intramuscularly. As a result, the plasmid DNA is taken up by the cells around the injection site, the expression of the gene incorporated in the eukaryotic expression unit on the plasmid occurs, and the gene product is produced. Although the direct injection method of DNA is currently mainly used for intramuscular injection, intramuscular injection (Yang JP et al., Gene. Ther. 3: 542-548, 1996
), Intradermal (Hengge UR et al., J. Clin. Invest. 97:
2911-2916, 1996; Choate KA et al., Hum. Gene.Th
er. 8: 1659-1665, 1997), brain (Schwartz B et al., Ge
ne Ther. 3: 405-411, 1996), gastric mucosa (Takehara T et
al., Hum. Gene. Ther. 7: 589-593, 1996).

The plasmid DNA to be injected intramuscularly is, for example, 1
μg to 1 mg, preferably 25 μg to 100 μg of DNA are dissolved in 50 μl of physiological saline and injected. This allows to get the highest value of expression after 4-7 days. For example, the gene encoding the c '' subunit is injected into plasmid pCMX.

(2-3) Anti-tumor treatment with antisense oligomers
Oligomers (phosphorothioate type oligomers) are often used. That is, antisense (MA) for the translation initiation region of the protein is prepared as an S-oligomer, and added to the culture solution of each cell to a final concentration of 10 μM. In an in vivo system using mice,
A method of injecting an antisense oligomer directly into a transplanted tumor is used. The optimal volumes at this time are different from each other, and the amount is determined with reference to the antitumor activity in an in vitro system.

10. Detection and Treatment of Cancer Using Antibodies In the present invention, an antitumor agent, a radioisotope, or the like is bound to a tumor-specific antibody, whereby cancer can be treated by so-called missile therapy, or cancer can be detected. When an antibody to which a radioisotope is bound is administered to a human or animal, the antibody assembles into a cancer lesion, so that the position of the cancer lesion can be known by detecting the radioisotope. This detection can be performed by imaging with a scintillation counter or a scintillation camera.

When performing missile therapy, an antitumor agent or a radioisotope capable of destroying cancer cells or cancer tissues is bound to an antibody, and the antibody is administered to the body. Examples of the radioisotope include ¹³¹ I and the like. Also,
Antitumor agents to be bound to the antibody of the present invention include, for example, cyclophosphamide, busulfan, alkylating agents such as nitrogen mustard, methotrexate, 5-FU,
Antimetabolites such as Ara-C, antibiotics such as actinomycin D, adriamycin, MMC, plant alkaloids such as vinblastine, vincristine, and VP-16, hormonal drugs such as estrogen, androgen, progesterone, and other cisplatin. Can be When ¹³¹ I is bound to an antibody, the antibody accumulates in cancer lesions and ¹³¹ I accumulates in the thyroid gland.

11. Method for detecting tumor-specific antigen using antibody, and reagent and kit for detection In the present invention, the purified antibody and the measurement target (blood,
By reacting with bodily fluids such as saliva and tears or tumor tissues), tumor cell-specific antigens can be detected and used for cancer diagnosis. Further, the purified antibody can be used as a reagent or kit for detecting a tumor-specific antigen.

The detection of the tumor antigen contained in the measurement object is performed by
This can be done by ELISA. First, a cancer antigen in a measurement target is adsorbed to each well of an ELISA multi-well plate (for example, 96 wells). After blocking with 1% BSA-PBS, an antibody (polyclonal antibody or monoclonal antibody) is reacted. Next, a biotinylated goat anti-mouse Ig is reacted. PB
After washing with S, a complex of biotinylated peroxidase and avidin was reacted (ABC method), and diaminobenzidine (0.
5mg / ml in 0.05M Tris-HCl pH7.5) and 3.1% hydrogen peroxide (H
₂ O ₂ ) is added to develop color. And the absorbance at 490nm
Measure with an ELISA reader.

The detection of tumor antigens can also be carried out using ECL immunoassay (Electrochemiluminescence Immunoassay) (Yusaburo Namba, Toshihito Kanajima; electrochemical immunoassay, clinical test: 293-300, 1998). In the present invention, the purified antibody is used alone or in an appropriate combination of a biotinylated goat anti-mouse Ig, a buffer such as PBS, a complex of biotinylated peroxidase and avidin, diaminobenzidine, hydrogen peroxide and the like. ,
It is used as a reagent or kit for detecting a tumor.

12. Method for detecting tumor-specific antibody using tumor-specific antigen peptide, and reagent and kit for detection In the present invention, the purified antigen peptide is reacted with a measurement target (a body fluid such as blood, saliva, tear fluid or a tumor tissue). Thereby, a tumor cell-specific antibody can be detected and used for cancer diagnosis.

The detection of a tumor-specific antibody contained in the measurement object can be performed by ELISA. First, each well of the 96-well plate for ELISA was used for the peptide of the present invention (M1, Ala10, Map
10, the protein described in SEQ ID NO: 2, the protein described in SEQ ID NO: 5, the protein described in SEQ ID NO: 8, the protein described in SEQ ID NO: 11, the protein described in SEQ ID NO: 14, or a partial sequence thereof). After blocking with% BSA-PBS, the measurement target is reacted. After washing with PBS, for example, biotinylated goat anti-mouse IgG, IgM or biotinylated rabbit anti-human IgG, IgM is reacted. Next, a complex of biotinylated peroxidase and avidin was reacted (ABC method), and diaminobenzidine (0.5 mg / ml in 0.05M Tr
is-HCl pH7.5) and 3.1% hydrogen peroxide (H ₂ O ₂ ) are added to develop color. Then, the absorbance at 490 nm is measured with an ELISA reader. The detection of the tumor-specific antibody can also be performed using an ECL immunoassay (Electro chemiluminescence Immunoassay) method or the like.

In the present invention, the purified peptide is
Used alone or as a reagent or kit for detecting antibodies, appropriately combining biotinylated goat anti-mouse Ig, a buffer such as PBS, a complex of biotinylated peroxidase and avidin, diaminobenzidine, hydrogen peroxide, etc. Is done.

13. Reagents and Kits for Tumor Gene Diagnosis and Tumor Detection Using Tumor-Specific Antigen Gene of the Present Invention The present invention utilizes the fact that tumor antigens are highly expressed in tumors and encodes the tumor-specific antigen protein DN
Genetic diagnosis of various tumors using A or RNA can be performed. The method of the present invention is basically characterized by reacting a tumor-specific antigen gene with a sample (tumor tissue or body fluid), and employs the following various methods. There are various methods for genetic diagnosis, and basically, for example, a PCR method for detecting RNA and a Northern method. Further, nested PCR and Taqman System are examples of improved methods used to increase the sensitivity and specificity of PCR.

In the nested PCR method, after amplification was performed using primers designed based on the sequence of a certain region of the target gene, the amplified fragment was designed based on the sequence of a region further inside the region. This is a technique of amplifying using primers, and can obtain high specificity and sensitivity.
The Taqman System designs a probe that hybridizes within the region to be amplified by PCR.The 5 'side of the probe is labeled with a reporter fluorescent dye, and the 3' side is labeled with a quencher dye that cancels the fluorescence. PCR using probe
It is a method of performing. When PCR is performed, the reaction by the polymerase proceeds, the probe is decomposed by the nuclease activity, the reporter dye is separated from the quencher dye, and the fluorescence is detected.

Further, for the detection of the gene, the NASBA method (Nuclei
c Acid Sequence Based Amplifiction). This method is sensitive and major-
It can detect bcr mRNA and is effective for early diagnosis of CML (chronic myeloid leukemia) and detection of minimal residual leukemia cells. That is, first, based on the sequence of the target gene,
Two types of primers (primer 1 and primer 2)
To design. Primer 1 has a T7 RNA polymerase promoter sense sequence on the 5 ′ side and a sequence complementary to the target RNA on the 3 ′ side. Primer 2 has a sequence complementary to the cDNA formed by priming Primer 1. First, the primer 1 is annealed to the target RNA, and then the 1st DNA is synthesized by AMV-RT (AMV reverse transcription). The resulting target RNA of the target RNA / cDNA hybrid is degraded by RNase H into single-stranded DNA. this
Anneal primer 2 to single-stranded DNA, template and T7 RNA
The promoter region of the polymerase becomes double-stranded. Then, T7 RNA polymerase recognizes this promoter sequence and synthesizes many copies of the antisense strand (RNA sequence) for the target RNA up to the 5 ′ end of primer 2. Primer 2 anneals to the newly synthesized antisense RNA,
CDNA (sense) is synthesized by AMV-RT. This RNA /
The RNA of the cDNA hybrid is degraded by RNase H and cD
NA (sense) is single-stranded. Primer 1 anneals to the 3 'end of the single-stranded cDNA, which induces the production of many RNA (antisense) copies. RNA amplified by the NASBA method is detected by a sandwich hybridization method.

Further, DN extracted from tumor tissue (cells)
A and DNA obtained from normal tissues (cells) are labeled with FITC and RITC, respectively, and hybridized with a normal chromosome sample (metaphase) in the presence of Cot1 DNA (blocking DNA),
There is a CGH (comparative genomic hybridization) method that determines the increase or decrease in copy number based on the fluorescence ratio of both (FITC / RITC).

In the DNA chip method, DNA of a patient to which a labeling substance has been bound is poured onto 1,000 or more copies of DNA that binds complementarily to the target DNA on a 1 cm square cover glass for microscope. This is a method for detecting a target DNA. This method uses a DNA complementary to the patient's DNA on the chip.
Only when NA is present, the patient's DNA binds, and the color develops due to the binding of the dye, and it is clear what kind of gene is expressed by which part of the chip has developed color, useful for tumor diagnosis It is. There is also a Western method for detecting proteins. Examples include methods using, for example, a tumor tissue diagnosis method (RT-PCR method) (FIGS. 14 to 1).
6, Figure 18, Figure 19).

14. Pharmaceutical composition comprising leukocytes reactive with the tumor-specific antigen peptide of the present invention or leukocytes binding to an antibody against the peptide as an active ingredient, and preventive, diagnostic or therapeutic agents for tumors Pharmaceutical composition comprising leukocytes of the present invention as an active ingredient The agent for preventing, diagnosing or treating a substance and a tumor may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Gum arabic, casein, gelatin, agar, glycerin,
Propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, etc., and artificial cell structures such as liposomes Can be The additives to be used are appropriately or in combination selected from the above according to the dosage form of the present invention.

When the leukocytes of the present invention are administered, parenteral administration such as intravenous injection, intratumoral injection, subcutaneous injection, intraperitoneal administration, intratumoral administration and the like can be employed. Further, the dose varies depending on the age, sex, administration route, number of administrations and the like of the administration subject, and can be changed in a wide range. In this case, the effective amount (the number of effective white blood cells) administered as a combination of the effective amount of the leukocyte of the present invention and a suitable diluent and a pharmacologically usable carrier is 1 × 10 ³ to 1 × 10 ^9.
/ kg body weight / day, given once or several times a day for more than one day.

When the leukocytes of the present invention are administered parenterally, additives such as stabilizers, buffers, preservatives, and isotonic agents can be contained, and they are prepared as needed. in this case,
After blood is collected from the patient and the target leukocyte is screened, the blood may be immediately returned to the patient.
(10 days, preferably 7 days) It is preferable to increase the degree of leukocyte activation by culturing. For example, there is a method in which leukocytes are cultured in the presence of a cytokine such as interleukin 2 to induce cytotoxic lymphocytes (CTL).

When the leukocytes of the present invention are used for diagnosing tumors, the CTLs and dendritic cells are separated and identified by a flow cytometer using a complex obtained by binding a fluorescent dye to the peptide from the blood to identify the tumors. Can be determined. Alternatively, coexistence of the peptide or peptide-presenting cell with leukocytes in the blood can be tested for an increase in nucleic acid synthesis to indicate the presence of a tumor.

When the leukocyte of the present invention is used as a diagnostic, prophylactic or therapeutic agent for tumor, the subject to be used is not particularly limited. For example, diagnosis, treatment or prevention of at least one kind of tumor such as cancer, sarcoma, and benign tumor can be used for a specific purpose. These diseases are
The leukocytes of the present invention can be used alone, in combination, or in combination with other diseases other than those described above.

Here, when the leukocytes of the present invention are used for the diagnosis, prevention or treatment of cancer, these cancer types are not particularly limited. For example, thyroid cancer, breast cancer, gastric cancer, esophageal cancer,
Oral, colon, rectal, anal, pancreatic, lung, renal, bladder, ovarian, uterine, vulvar, skin, melanoma, central or peripheral nerve tumors (meningioma, glioma , Auditory nerve tumors, brain, spinal cord, and peripheral nerve tumors including neurofibrosarcoma), gingival cancer, pharyngeal cancer, jaw cancer (including maxillary cancer; squamous cell carcinoma), mediastinal tumor (including thymic cancer), liver cancer, bile duct Cancer, gallbladder cancer, renal pelvic tumor, ureteral cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma (uterine carcinosarcoma) At least one selected from the group consisting of: However, not only a single cancer but also a combination of multiple cancers is included.

15. Functional composition (antitumor food etc.) Since the peptide of the present invention can be administered orally, it can also be used as a functional composition, particularly a functional antitumor food or a functional antitumor drug. In this case, the peptide of the present invention can be administered as it is or as it is contained in an enteric capsule. Alternatively, it may be dissolved in a liquid (preferably water) so as to have an appropriate concentration, and added to the food by a method such as mixing, dipping, coating, spraying, or the like. As a result, the present peptide can be used as a functional food such as meat, fish, and vegetables. When the peptide of the present invention is used as an aqueous solution, 0.001 mg to 1 mg, preferably 0.01 to 0.5 mg
It can be.

The Pr, which is an epitope of the tumor-specific antigen,
o When the database was searched by the alignment method using the amino acid sequence of Phe Ser Ala Thr Asp Pro Lys Ala, it was part of aquaporin, a water channel protein that controls the access of water to various plants. (SEQ ID NO: 4
6-52). [Pinus strobus], [Pinus bank]
siana], [Oryza sativa], [Arabidopsis thaliana], [P
icea mariana], [Samanea saman], [Mesembryanthemum]
crystallinum]. Pro Phe Ser Ala Thr A
Aquaporins containing sp Pro Lys Ala can be efficiently extracted and concentrated from plants and orally administered as fractions, which can be used as food substitutes for tumor vaccines. As a method for efficiently extracting aquaporin from plants, far-infrared irradiation, fermentation, roasting, and other methods are used in combination with cellulase, proteolytic enzyme, and the like.

16. Pharmaceutical compositions containing various proton pump inhibitors of the present invention as active ingredients and preventive or therapeutic agents for tumors Pharmaceutical compositions containing various proton pump inhibitors of the present invention as active ingredients and preventive or therapeutic agents for tumors include: It may contain a pharmaceutically acceptable carrier or additive together. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol,
In addition to stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, artificial cell structures such as liposomes and the like can be mentioned. The additives to be used are appropriately or in combination selected from the above according to the dosage form of the present invention.

In the present invention, a prophylactic or therapeutic agent for tumors containing a proton pump inhibitor can be administered orally or parenterally. When administering the preventive or therapeutic agent of the present invention orally, tablets, granules,
Solid preparations such as powders and pills, or liquid preparations such as liquid preparations and syrup preparations may be used. In particular, granules and powders can be in unit dosage form as capsules, or in the case of liquid preparations as dry products to be re-dissolved when used. Of these dosage forms, oral solids are
The compositions usually contain additives such as binders, excipients, lubricants, disintegrants, wetting agents and the like which are generally used in pharmaceutical preparations. In addition, oral liquid preparations are usually used in those compositions in the formulation generally used stabilizers, buffers, flavoring agents,
Contains additives such as preservatives, fragrances and coloring agents.

When the prophylactic or therapeutic agent of the present invention is administered parenterally, injections, suppositories and the like may be used. In the case of injection, it may be provided in the form of a unit dose ampoule or a multi-dose container, and may be a powder reconstituted with a suitable carrier for use, for example, sterile pyrogen-free water. These dosage forms usually contain additives, such as emulsifiers and suspending agents, which are generally used in pharmaceutical compositions. Injection techniques include, for example, intravenous infusion, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intradermal injection, and intratumoral injection. The dose varies depending on the age of the subject, the route of administration, and the number of administrations, and can be varied over a wide range.

In this case, the effective amount administered as a combination of the effective amount of the prophylactic or therapeutic agent of the present invention and a suitable diluent and a pharmacologically usable carrier is, for example, the case of EDTA (chelation therapy). Is administered as a single dose of 50 mg / kg body weight x creatinine clearance / 100 EDTA by infusion over 3-4 hours, 2-4 times a week, paying attention to renal function. Other drugs should be administered in compliance with their respective doses. The proton pump inhibitor used as a prophylactic or therapeutic agent for tumors of the present invention includes, for example, the following, and one type can be used alone or two or more types can be used in combination.

(1) V-ATPase inhibitor 1. EDTA (Ethylenediaminetet
raacetic acid), 2. Bafilomycin A1, Bafilomycin B1 (Droese S et al., Biochemistry. 32: 3902-3906, 199
3; Bowman EJ et al., Proc. Natl. Acad. Sci. USA 8
5: 7972-7976, 1988), 3. EGTA (ethylene glycol bis (2-aminoethyl ether) -N, N, N ', N'-tetraacetic acid) [Ethylene Glycol bis (2-aminoethyl ether) -N, N , N ',
N'-tetraacetic acid], 4. Concanamycin A, 5. Concanamycin B, 6. O-methylconcanamycin B
B) (Someno K et al., J. Antibiot. 37: 1333-1343, 198
4; Kinashi H et al., JAntibiot. (Tokyo) 35: 1618-
1620, 1982), 7.N-ethylmaleimide (NEM), 8.N, N'-dicyclohexylcarbodiimide (N, N'-dicyc
lohexylcarbodiimide), 9. Leucanicidin (Isogai A et al., Biol. Chem. 48: 1379-1381, 198
4), 10. 7-chloro-4-nitrobenz (o) -2-oxa-1,3-diazole [NBD-Cl] (7-chloro-4-nitrobenz (o) -2-oxa-1,3- diazole) [NBD-C
l], 11. Suramin, 12. Verapamil (VPL), 13. (2Z, 4E) -5- (5,6-dichloro-2-indolyl) -2-methoxy-N- (1 , 2,2,6,6-pentamethylpiperidin-4-yl) -2,4
-Pentadienamide ((2Z, 4E) -5- (5,6-dichloro-2-indolyl) -2-methoxy-N-
(1,2,2,6,6-pentamethylpiperidin-4-yl) -2,4-pentadie
namide (Nadler G et al., Bioorg. Med. Chem. Lett. 8: 362
1-3626, 1998), 14.Destruxin B, 15.Destruxin E (Muroi M et al., Biochem. Biophys. Res. Commun. 2
05: 1358-1365, 1994; Naganuma S et al., Biochim. B
iophys. Acta. 1126: 41-48, 1992; Steiner J Ret a
l., Int. J. Pept. Protein. Res. 31: 212-219, 1988;
Pais M et al., Phytochemistry. (Oxford) 20: 715-7
24, 1981), 16.OST-766 (Morris SA et al., Calcif. Tissue. Int. 64: 148-1
53, 1999), 17.5- (5,6-dichloro-2-indolyl) -2-methoxy-2,4-
Pentadienamide (5- (5,6-Dichloro-2-indolyl) -2-methoxy-2,4-pentadi
enamides) (Gagliardi S et al., J. Med. Chem. 41: 1568-1573,
1998), 18.NO ₃ (Chow CW et al., J. Gen. Physiol. 110: 185-200, 1
997), 19.3'-O- (4-benzoyl) benzoyladenosine 5'-triphosphate
) (BzATP) (Manolson MF et al., J. Biol. Chem. 260: 12273-12
279, 1985; Vasilyeva Eet al., J. Biol. Chem. 271:
12775-12782, 1996), 20. Hygrolidin (Seto H et al., Tetrahedron. Lett. 23: 2667-2670,
1982), 21. Bisphosphonate tiludronate
e tiludronate) (David Pet al., J. Bone. Miner. Res. 11: 1498-150
7, 1996), 22. Diethylpyrocarbonat
e) (DEPC), 23. Dibromoacetophenone, 24. p-Chloromercuribenzoic a
cid), 25. quercetin, 26. 4-acetamido-4'-isothiocyanostilbene-2,
2'-Disulfonic acid (4-acetoamide-4'-isothiocyanostilben-2,2'-disylfo
nic acid (SITS)), 27. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
(DIDS), 28. N-Bromosuccinimide, 29. Benzoyl-taxinine-K

(2) H + / K + -ATPase inhibitor (2-1) Benzimidazole derivative 1. Sodium rabeprazole, 2.2-[[4- (3-methoxypropoxy) -3-methylpyridine
-2-yl] methylsulfinyl] -1H-benzimidazole ((±) -sodium 2-[[4- (3-methoxypropoxy) -3-methylpyri
din-2-yl] methylsulfinyl] -1H-benzimidazole), 3. Omeprazole (±) -5-methoxy-2- [[(4-methoxy-3,5-dimethyl-2-
Pyridyl) methyl] -sulfinyl] benzimidazole ((±) -5-Methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyr
idyl) methyl] -sulfinyl] benzimidazole), 4. Pantoprazole (±) -sodium-5-difluoromethoxy-2-[[(3,4-dimethoxy-2-pyridinyl) methyl] -sulfinyl] -1H -Benzimidazole ((±) -Sodium-5-difluoromethoxy-2-[[((3,4-dimethoxy
-2-pyridinyl) methyl] -sulfinyl] -1H-benzimidazole
), 5. Leminoprazole (±) -2-[[2- (isobutylmethylamino) benzyl] sulfinyl] -1H-benzimidazole ((±) -2-[[2- (isobutylmethylamino) benzyl]) sulfiny
l] -1H-benzimidazole) (Ishihara K et al., Arzneimi
ttelforschung.44: 827-830, 1994), 6.2-Dimethylamino-4,5-dihydrothiazolo [4,5: 3,
4] pyridol [1,2-a] benzimidazole 2-dimethylamino-4,5-dihydrothiazolo [4,5: 3,4] pyrido
l [1,2-] a benzimidazole (Chung YK et al., Can. J. Physiol. Pharmacol. 76:
921-929, 1998), 7. 2-Aryl-4,5-dihydro-1H-thieno [3,2-e] benzimidazole 2-aryl-4,5-dihydro-1H-thieno [3,2-e ] benzimidazole (Homma K et al., Chem. Pharm. Bull. (Tokyo) 45: 19
45-1954, 1997), (2-2) Other than benzimidazole derivatives 1. (±) -5-methoxy-2-[[(4-methoxy-3,5-dimethylpyrid-2-yl) methyl] sulfinyl]- 1H-imidazo (4,5-
b) Pyridine (±) -5-methoxy-2-[[(4-methoxy-3,5-dimethylpyrid-2-
yl) methyl] sulphinyl] -1H-imidazo (4,5-b) pyridine (TU-
199) (Uchiyama K et al., J. Pharm. Pharmacol. 51: 457-6
4, 1999; Uchiyama K etal., Methods. Find. Exp. Cli
n. Pharmacol. 21: 115-122, 1999), 2. 3-Amino-5-methyl-2- (2-methyl-3-thienyl) imidazo [1,2-a] thieno [3,2-c] Pyridine 3-amino-5-methyl-2- (2-methyl-3-thienyl) imidazo [1,2
-a] thieno [3,2-c] pyridine (Ushiro T et al., Jpn. J. Pharmacol. 75: 303-306,
1997), 3. 1-aryl-3-substituted pyrrolo [3,2-c] quinoline (Yum E K., Bioorg. Med. Chem. Lett. .9: 2819-2822,
1999), 4. 3-butyryl-8-methoxy-4-[(2-thiophenyl) amino] quinoline 3-butyryl-8-methoxy-4-[(2-thiophenyl) amino] quinoli
ne (Kim K B., J. Pharm. Pharmacol. 50: 521-529, 199
8),

5. 2-[(3,5-dimethyl-4-methoxypyridyl) alkyl] -benzothiazolidine 2-[(3,5-dimethyl-4-methoxypyridyl) alkyl] -benzothia
zolidine (Yoon SH et al., Bioorg.Med.Chem. Lett. 8: 1909
-1912, 1998), 6. 3-butyryl-4- [R-1-methylbenzylamino] -8-ethoxy-1,7-naphthyridine 3-butyryl-4- [R-1-methylbenzylamino] -8-ethoxy -1,7-n
aphthyridine (Sohn SK et al., J. Pharm. Pharmacol. 51: 1359-13
65, 1999), 7. Pentagalloylglucos
e): 1,2,3,4,6, -penta-o-galloyl-β-D-glucose) (1,2,3,4,6, -penta-o-galloyl-beta-D-glucose) (Ono K et al., Clin. Chim. Acta. 290: 159-167, 200
0)

(3) H + / Cl- Symporter 1. Prodigiosin (PG) (Sato Tet al., J. Biol. Chem. 273: 21455-21462, 19)
98), 2. Metacycloprodigiosin
(M-PG), 3. Prodigiosin 25-C (PG25-C) (Harashima K et al., J. Agr. Biol. Chem. 31: 481-4
89, 1967), 4. Cycloprodigiosin, 5. Cycloprodigiosin hydrochloride
odigiosin hydrochloride) (cPrG.HCl) (Kawauchi K et al., Biochem. Biophys. Res. Commun.
237: 543-547, 1997), 6. Bepridil, 1-Isobutoxy-2-pyrrolidino-3- [N-benzylanilino] propane benzylan
ilino] propane), 7. Manganese tetraphenylporphine (5,10,15,20-tetraphenyl-21H, 23H-porphinemanganese (III) chloride) (5,10,15.20-Tetraphenyl-21H, 23H-porphine manganes
e (III) Chloride), 8. Thallium chloride (TlCl ₃ )

[0151]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. Example 1 Preparation of Monoclonal Antibody and Examination of Properties A: Extraction of Antigen from Cancer Cell Membrane Components
A long-term culture in -10% fetal calf serum established a cell line, designated TPC-1. This cell line was cultured in large quantities and extracted with the aim of extracting the protein portion of the cell membrane.
ith et al. (Smith JT, et al., Exp.Cell Res. 13:
96-102, 1957.) (Kurata Y.
and Okada S., Int.Arch.Allergy, 29: 495-509, 19
66.) was used to prepare an antigen as follows. That is, R
5 × 10 ⁷ cells obtained by culturing at 37 ° C. for 20 days in PMI1640 medium were crushed using solution I (0.16 M KCl, 1 mM monoiodoacetic acid, 17 mM sodium citrate), and
Centrifugation was performed at 10,000 xg for 30 minutes. The insoluble fraction was added to solution I
The suspension was suspended in I (1 M KCl, 1 mM monoiodoacetic acid, 34 mM sodium citrate) and centrifuged at 10,000 × g for 30 minutes at 4 ° C. This process was repeated several times, and the obtained precipitate was suspended in 0.25% sodium deoxycholate and stirred at 4 ° C for 48 hours. This was centrifuged at 10,000 × g for 30 minutes, 10 times the volume of cold acetone was added to the obtained supernatant, and the mixture was allowed to stand at −20 ° C. overnight. The resulting precipitate was centrifuged and dissolved in a small amount of 10 mM PBS (phosphate buffer). This is Blue Sepharose CL-6B
(Pharmacia) to remove mixed albumin.
The obtained eluted fraction was concentrated and air-dried.

B: Preparation of Hybridoma Preparation of the hybridoma was carried out according to a known method. That is, the extract obtained from the above membrane component was suspended in complete adjuvant, and subcutaneously injected into Balb / c mice at intervals of 2 weeks.
Injections. Two days after the final immunization, cells were separated from the spleen, and lymphocytes were removed by separating blood cells using a centrifuge. With polyethylene glycol, lymphocytes 5 × 10 ⁷ mouse myeloma cells (SP2 / 0) with obtained was 10 ⁷ fusion. Selection with the HAT medium was performed by changing the medium by half every three days and culturing the cells with the HT medium after the ninth day. As a result of screening by the limiting dilution method, many monoclonal antibody strains were obtained. In addition, the following test was performed to select cells which reacted with thyroid cancer cells but did not react with normal thyroid cells and other normal cells. That is, normal thyroid cells NTC-1, human fetal kidney cells HEK-1 and thyroid cancer cells TPC-
Spread 1 to 5 × 10 ⁵ pieces into the well of the vinyl plate,
Using poly-L-lysin (MW: 520,000), adhesion was performed at 4 ° C. for 2 hours. Then use 0.25% glutaraldehyde at room temperature
Cells were fixed for 15 minutes. Blocking is 1% BSA-PBS-0.2
Performed at 37 ° C. for 2 hours in the presence of 5% NaN ₃ . Next, each supernatant of the hybridoma was added and reacted at 37 ° C. for 1 hour, and peroxidase-linked anti-mouse Ig was allowed to act at 37 ° C. for 1 hour. Peroxidase activity was expressed using OPD (O-phenylenediamine; Sigma) and 0.03% H ₂ O ₂ and 15 minutes later an ELISA reader (Bio-Tec. Instrument In).
c. USA) at 490 nm.

As a result, a hybridoma producing the monoclonal antibody of the present invention was obtained and named KTC-3. Hereinafter, the antibody produced by hybridoma KTC-3 is referred to as `` KTC
-3 antibody ". Hybridoma KTC-3 (name: hybridoma KTC-3), which produces KTC-3 antibody, is available from the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan).
No. FERM BP-6899, and deposited internationally under the Budapest Treaty (Original deposit date: September 30, 1998)
Day).

C. Monoclonal Antibody of the Invention (KTC-3 Antibody)
In this example, the cell ELISA method (Cell enzyme-linked imm
In order to confirm the cancer specificity of the KTC-3 antibody using unosorbent sandwich assay), the following experiments were performed on five normal cells including thyroid cells, six thyroid cancer cells, and cell lines derived from other cancers.

[0155] That is, poly-L-lysi 10 ⁵ each cell
n (MW: 520,000) to adhere to an ELISA plate at 4 ° C. for 2 hours. Next, the cells were fixed using 0.25% glutaraldehyde at room temperature for 15 minutes. Blocking is 1% BSA-PBS
The presence of -0.25% NaN _3, was carried out for 2 hours at 37 ° C.. Next, KTC-
Three antibodies were added and reacted at 37 ° C. for 1 hour. After washing, peroxidase-linked anti-mouse Ig was allowed to act for 1 hour at 37 ° C. The peroxidase activity was between OPD and 0.03% H ₂ O ₂
And after 15 minutes an ELISA reader (Bio-Tec.
nstrument Inc. USA) at 490 nm. As a result, the antibody of the present invention was specific to cancer cells (Table 1). In Table 1, NTC-1, TPC-1, TPC-2, TPC-3, TUC
-1 is a cell line established by the present inventors.

[0156]

[Table 1]

Further, it was confirmed by the fluorescent antibody method that the KTC-3 antibody reacted with thyroid cancer cells but did not react with normal thyroid cells (FIGS. 2 and 3). Next, using immunohistochemical staining,
It was confirmed that the KTC-3 antibody reacted with thyroid cancer (FIGS. 4 and 5). In FIG. 4, it can be seen that the KTC-3 antibody does not react with follicles formed by normal cells, but reacts strongly with cancer cells.

D. Confirmation of Location of Tumor Antigen in Cancer Cells In order to confirm where in the tumor cells the tumor-specific antigen recognized by the monoclonal antibody of the present invention is located, a method using colloidal gold particles is performed. Adopted. The KTC-3 antibody was reacted with TPC-1 and observed by immunoelectron microscopy. A KTC-3 antibody was used as a primary antibody, and an anti-mouse goat immunoglobulin antibody labeled with colloidal gold was used as a secondary antibody. That is, the cultured TPC-1 cells were treated in the following procedure.

Prefix: 4% paraformaldehyde + 0.1
% Glutaraldehyde + 0.15M cacodylate buffer,
4 ° C, 5 minutes Pellet: Approx. 200 × g, 5 minutes centrifugation (4 ° C) Treated with TBS containing 0.1M lysine for 30 minutes and centrifuged Dispersion: Disperse cells in TBS containing 0.5% BSA Primary antibody: BSA-TBS Antibody KTC-3, diluted to 1 ° C, overnight at 4 ° C Rinse: rinse 3 times with BSA-TBS Gold colloid: Colloidal gold-labeled anti-mouse goat immunoglobulin antibody, room temperature 30 minutes, rinse 3 times with TBS

Fixation: 2% glutaraldehyde in cacodylate buffer (pH 7.4), 4 ° C., 3 minutes. Rinse with TBS 1
Times. Post fixation: 2% osmate in cacodylate buffer, 4 ° C, 60 minutes. Rinse with cacodylate buffer. Dehydration, embedding: ethanol dehydration, Epon embedding As a result, colloidal gold particles were observed on the cancer cell membrane (Fig. 6, wedge-shaped arrow). Therefore, it was shown that the tumor antigen reactive to the KTC-3 antibody was present on the surface of the cancer cell membrane (x 65,000).

E. Demonstration of Tumor Antigen as Protein The tumor antigen extracted from the cell membrane of TPC-1 was subjected to various chemical pretreatments on an ELISA plate, and then reacted with the KTC-3 antibody. It was measured (Table 2). The color development of the tumor antigen was significantly reduced by the treatment with the proteases trypsin and pronase E, compared to the color development without treatment (none). However, treatment on sugar was unaffected. This indicates that this antigen is a protein.

[0162]

[Table 2]

F. Cell Killing Effect of KTC-3 Antibody on Thyroid Cancer Cells (1) Effect of KTC-3 Antibody The effect of KTC-3 antibody on human thyroid cancer cell line TPC-1 was examined. The KTC-3 antibody was added to the TPC-1 cell culture system, and TPC-1 cells (from human thyroid cancer) were observed 72 hours later (FIG. 7).

That is, thyroid cancer cells TPC-1 and hybridoma KTC-3 were each cultured. The culture solution is RPM
I1640-10% fetal calf serum was used. TPC-1 after 24 hours of culture
Was replaced with the culture supernatant of the hybridoma. Observation was started from this point. As a result, after 72 hours, all cancer cells had died with trypan blue staining.

(2) Cell-killing effect of the KTC-3 antibody on the thyroid cancer cells of the Fab part The KTC-3 antibody was separated into the Fab part and the Fc part by papain treatment, and the Fab part was separated using a Protein A Sepharose column. (FIG. 8). First, Hybridoma K
The culture supernatant of TC-3 was collected, saturated ammonium sulfate was added to make it 45% saturated, and the protein was precipitated. This was collected in a dialysis tube and desulfurized with a large amount of water. Next, Sepharose CL-6
Gel filtration was performed using B to obtain the antibody KTC-3 at the first peak. Then, affinity chromatography using protein A was performed. That is, IgGSepharose 6 Fast
Using Flow (Pharmacia), 50 mM Tris buffer, pH
Elute the Fab portion with 7.6,150 mM NaCl, then 0.5M acetic acid pH 3.4
(Adjusted with ammonium acetate) to elute the Fc portion.

The TPC-1 of the Fab portion obtained as described above
The effect on was investigated. That is, a culture solution (RPMI1) was prepared for Fab portion 1 obtained by affinity chromatography.
640-10% fetal calf serum) 9. This was previously cultured in a culture solution of TPC-1 cells (RPMI1640
-10% fetal calf serum). After 72 hours, the presence or absence of trypan blue staining positive cells (dead cells) was observed. The results are shown in FIGS. At 24 hours after the addition of the Fab portion, there are few dead cells stained with trypan blue (Fig.
9) After 72 hours, all cells had died with trypan blue staining positive (FIG. 10).

Summary 1 1. The KTC-3 antibody reacted cancer-specifically with cells derived from thyroid, gastric, colon and kidney cancers. 2. The tumor antigen recognized by the KTC-3 antibody was present on the cancer cell membrane. 3. The tumor antigen recognized by the KTC-3 antibody was protein, not sugar. 4. The KTC-3 antibody worked cytocidally against thyroid cancer. A similar effect was seen with the Fab portion alone.

Example 2 Analysis of Tumor Antigen Gene (1) Cloning of cDNA Encoding Tumor Antigen Protein Thyroid Cancer Cell Line TPC Using λgt11 Expression Vector
-1 cDNA library was prepared. That is, the GTC / Li method (Cathala et al., 1983, DNA 2, 329-3) was performed on TPC-1 cells.
Total RNA was extracted by 35), and poly (A) + RNA was purified by oligo (dT) column chromatography. Next, using 5 μg of the poly (A) + RNA as a template, double-stranded cDNA was synthesized using a cDNA synthesis system plus of Amersham. Further, using 1 μg of the cDNA and an Amersham cDNA cloning system λgt11,
Create a library and use Stratagene's GIGAPACK II Go
Packaged using ld. The reaction conditions were Amersham
And Stratagene protocols were followed.

The library obtained as described above was screened using the KTC-3 antibody. And K
A clone reacting with the TC-3 antibody was subcloned into the plasmid pRIT 2T, and the dideoxy method (Sanger F., Nicklen
S, Coulson A R .: Proc. Natl. Acad. Sci. 74: 5463-5
467, 1977).

As a result, a partial fragment of the gene encoding the thyroid cancer tumor antigen protein was obtained, and its sequence was represented by the nucleotide sequence of positions 337 to 986 of the nucleotide sequence shown in SEQ ID NO: 1). there were. The sequence of the protein (tumor antigen protein) encoded by the nucleotide sequence was represented by amino acids 86 to 205 of the amino acid sequence of SEQ ID NO: 2. Then, this tumor antigen protein of thyroid cancer was named S-1 tumor antigen.

(2) cD encoding S-1 tumor antigen protein
Isolation of the 5 'region of NA and determination of total nucleotide sequence After extracting total RNA from TPC-1 cells by the GTC / Li method, oligo
Poly (A) + RNA was purified by o (dT) column chromatography. Next, using 0.5 μg of the poly (A) + RNA as a template
5 'of S-1 tumor antigen cDNA using BRL's 5'-RACE system
The region was amplified by PCR and isolated. That is, a single-stranded cDNA was synthesized by a reverse transcriptase using the gene-specific oligo DNA (GSP1) designed in the region where the sequence had already been determined as a primer. Next, TdT was added to the 3 'end of the cDNA.
(Terminal deoxynucleotidyl transferase) and dCTP added the dC polymer. Using it as a template, an adapter primer with dG polymer and GS
The first PCR was performed using GSP2 designed upstream of P1 and a primer. Furthermore, a second PCR was performed using the adapter primer and GSP3 designed further upstream.
The gene fragment obtained as described above was
It was subcloned into script II and its nucleotide sequence was determined by the dideoxy method. The sequence was represented by the 1st to 336th sequence of the nucleotide sequence described in SEQ ID NO: 1.
The sequence of the protein encoded by the nucleotide sequence was represented by amino acids 1 to 85 of the amino acid sequence described in SEQ ID NO: 2. As described above, the entire nucleotide sequence and the entire amino acid sequence of the cDNA encoding the thyroid cancer tumor antigen protein were successfully determined. The thyroid cancer tumor antigen protein was named SSY tumor antigen.

(3) Search for amino acid sequence homology Next, a database was searched by the alignment method using the amino acid sequence of the protein obtained in the above (2). As a result, the amino acid sequence of the thyroid cancer SSY tumor antigen was determined by the amino acid sequence of the proteolipid subunit (ATP6F, hereinafter referred to as c '') of vacuum H + -ATPase (proton pump) reported by Nishigori et al. In June 1998. The sequence was found to be identical (Nishigori H, et al, Genomics. 1998 Jun 1; 50
(2): 222-228). In addition, SSY tumor antigen is PCT
/ JP97 / 04056 (WO 98/21328).

(4) Determination of Antigenic Determinant (Epitope) Recognized by KTC-3 Antibody The amino acid sequence of the S-1 tumor antigen was determined by the N14 profile method (hydr
As a result of analysis using ophobicity plotting, amino acid residue 1
16 to 125 (116 to 125 of the amino acid sequence of SEQ ID NO: 2)
) Was estimated to protrude outside the membrane.
Therefore, this part was expected to be an antigenic determinant (epitope) recognized by the KTC-3 antibody.

Therefore, amino acid residues 116 to 125 were synthesized, and amino acid residues 102 to 111 (Ile Tyr Gly Il
e Ile Met Ala Ile Val Ile: SEQ ID NO: 20)
The reactivity with the -3 antibody was examined. The reactivity was tested in the same manner as in the ELISA described in Example 1, except that a nitrocellulose membrane was used instead of the ELISA plate. As a result, the antibody reacted only with amino acid residues 116 to 125 (FIG. 11). Furthermore, peptides were sequentially removed one by one from the N-terminal side and the C-terminal side, and the reaction with the KTC-3 antibody was examined.

As a result, amino acid residues 118 to 124 (Phe Se
r Ala Thr Asp Pro Lys)
Does not react with KTC-3 antibody and eventually amino acid residues 118-12
4 (Phe Ser Ala Thr Asp Pro Lys) was found to be an epitope of the KTC-3 antibody (FIG. 12). We named this epitope the M-1 peptide.

Further, the amino acid sequence of the SSY tumor antigen was
Analysis by phobicity plotting revealed that amino acid residue 116
~ 148 (Phe Ser Ala Thr Asp Pro Lys Ala Ile Gly His
ArgAsn Tyr His Ala Gly Tyr Ser Met Phe Gly Ala Gl
y Leu Thr Val; 116th amino acid sequence of SEQ ID NO: 2
148148th sequence) (SEQ ID NO: 21) was estimated to project outside the membrane (FIG. 13). So this amino acid residue
Synthesized 116-148 and examined the reactivity with KTC-3 antibody,
Finally, amino acid residues 118 to 124 (Phe Ser Ala Thr Asp
Pro Lys) was reconfirmed to be an epitope of the KTC-3 antibody.

(5) Expression of S-1 tumor antigen in normal cells and various tumor cells Nishigori H et al. Determined that c ″ (ATP6F:
(Fig. 1) has been reported to be universally expressed in human normal cells (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) (Nishigori H, et al., Genomics. Jun. 1; 50
(2): 222-228, 1998.), but does not mention tumor cells at all. Also, there is no description about the specific gene function of the sequence represented by SEQ ID NO: 1 described in PCT / JP97 / 04056 (WO 98/21328).

Therefore, the present inventors first examined the expression of the S-1 tumor antigen in various tumor tissues (removed surgical specimens) by RT-PCR, and then examined the expression of the SSY tumor antigen. RT-PC
R was carried out using a TOYOBO RT-PCR high kit according to the instructions. That is, all RNs extracted from various tumor tissues
A PCR was performed using cDNA obtained by reverse transcription of 25 ng of A as a template. PCR was performed at 94 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C.
This was performed 33 cycles with the condition of 1 minute at 1 ° C. as one cycle. The following primers were used for the S-1 tumor antigen and the SSY tumor antigen.

Forward primer (F1 to F4 primer) F1 primer (forward primer 1): 5'-CAAGAACCTGGTCA
GCATCATC-3 '(SEQ ID NO: 22) (corresponding to the 345th to 366th sequence of the nucleotide sequence described in SEQ ID NO: 1) F2 primer (forward primer 2): 5'-TGTGAGGCTGTGGC
CATCTA-3 ′ (SEQ ID NO: 23) (SEQ ID NO: 1)
F3 primer (forward primer 3): 5'-AGACTGCGGGACG-
3 '(SEQ ID NO: 24) (corresponding to the first to 13th sequences of the nucleotide sequence described in SEQ ID NO: 1) F4 primer (forward primer 4): 5'-CGCCATGACGGGGC
TA-3 '(SEQ ID NO: 25) (78th to 78th of the nucleotide sequence described in SEQ ID NO: 1)
(Corresponds to the 93rd sequence)

Reverse primer (R1 to R3 primer) R1 primer (reverse primer 1): 5'-CCCCACCCACACAT
ATCATC-3 ′ (SEQ ID NO: 27) (SEQ ID NO: 1)
R2 primer (reverse primer 2): 5'-AATGGTGTAGCAGA
CTCC-3 ′ (SEQ ID NO: 28) (No. 14 of the nucleotide sequence of SEQ ID NO: 1)
R3 primer (reverse primer 3): 5'-CATGGAGTAGCCTG
CATGG-3 ′ (SEQ ID NO: 29) (SEQ ID NO: 4
(Sequence complementary to the 74th to 492nd sequence)

[0181] DN amplified by these primers
The size of the A fragment is, for example, 374 bp when the F1 and R1 primers are used, and 349 bp when the F2 and R1 primers are used. As a positive control, a primer (included in the kit) for a housekeeping gene (glyceraldehyde triphosphate dehydrogenase (GAPDH)) was used. The size of the DNA fragment amplified by these primers is 450 bp. When no reverse transcription reaction was performed (-RT), it was confirmed that no band appeared (FIG. 15, lane 4, lane 4).
8, 13, 17).

After the PCR reaction, DNA fragments were confirmed by agarose gel electrophoresis. The size marker is a 100 bp ladder (Pharmacia). As a result, in humans, breast cancer (adenocarcinoma), gastric cancer (adenocarcinoma), esophageal cancer (adenocarcinoma, squamous cell carcinoma), oral cancer (squamous cell carcinoma), lung cancer (adenocarcinoma, squamous cell carcinoma), ovarian cancer (glandular carcinoma) Cancer), endometrial cancer (adenocarcinoma), vulvar cancer (squamous cell carcinoma), melanoma, liver cancer (adenocarcinoma), and malignant lymphoma. Strong expression of S-1 tumor antigen and SSY tumor antigen was detected. However, in the normal tissue corresponding to each organ of the same patient used as a control, very slight or almost no detection was found (FIGS. 14, 15 and 16).

In FIGS. 14 to 16, the contents of the samples in each lane are shown in Table 3 for FIG. 14, and in Table 4 for FIG.
FIG. 16 is shown in Table 5.

[0184]

[Table 3]

[0185]

[Table 4]

[0186]

[Table 5]

Summary 2 1. Preparation of cDNA library of thyroid cancer cell line TPC-1,
Tumor antigen protein that reacts with KTC-3 antibody (S-1 tumor antigen)
Was identified. Furthermore, the entire amino acid sequence of the tumor antigen protein was determined (SSY tumor antigen). The amino acid sequence of the SSY tumor antigen is based on the proteolipid subunit ATP6F (c ″ prime) of vacuum H + -ATPase (proton pump) (Nishigo
ri, H. et al., GENOMICS 50, 222-228 (1998)). 2. The antigenic determinant (epitope) recognized by the KTC-3 antibody
Phe Ser Ala Thr AspPro Lys (M-1 peptide) was revealed.

3. SSY tumor antigens include all cancers that we can test for, namely thyroid cancer (well differentiated adenocarcinoma, undifferentiated adenocarcinoma), breast cancer (adenocarcinoma), gastric cancer (adenocarcinoma), Esophageal cancer (adenocarcinoma, squamous cell carcinoma), oral cancer (squamous cell carcinoma), colorectal cancer (adenocarcinoma), rectal cancer (adenocarcinoma), anal cancer (squamous cell carcinoma), pancreatic cancer (adenocarcinoma), lung cancer ( Adenocarcinoma, squamous cell carcinoma, small cell carcinoma), renal carcinoma (adenocarcinoma), bladder carcinoma (transitional cell carcinoma), ovarian carcinoma (adenocarcinoma), endometrial carcinoma (adenocarcinoma), cervix carcinoma (adenocarcinoma) , Squamous cell carcinoma),
Vulvar cancer (squamous cell carcinoma), skin cancer (squamous cell carcinoma), melanoma, central or peripheral nerve tumor (meningioma, glioma, auditory nerve tumor, brain / spinal cord / peripheral nerve tumor including neurofibrosarcoma), gingiva Cancer (squamous cell carcinoma), pharyngeal cancer (squamous cell carcinoma),
Jaw cancer (including maxillary cancer; squamous cell carcinoma), mediastinal tumor (including thymic cancer), liver cancer (adenocarcinoma), bile duct cancer (adenocarcinoma), gallbladder cancer (adenocarcinoma), renal pelvis tumor (transitional cell carcinoma) , Ureteral cancer (transitional cell carcinoma), testicular tumor, prostate cancer (adenocarcinoma), choriocarcinoma, fallopian tube cancer (adenocarcinoma), vaginal cancer (squamous cell carcinoma), sarcoma (rhabdomyosarcoma,
Leiomyosarcoma, including osteosarcoma), leukemia, erythroleukemia, multiple myeloma, malignant lymphoma, and carcinosarcoma (including uterine carcinosarcoma). 4. Is SSY tumor antigen very weak in normal tissue,
Little was detected.

Example 2 Analysis of Gene Encoding Mouse Tumor Antigen In this example, erythroleukemia cell EL-4 was used to analyze genes expressing a tumor-specific antigen in mouse tumor cells. The tumor antigen gene in mice was analyzed by PCR. The RT-PCR method uses TOYOBO's RT-PCR high
This was performed using the kit according to the instructions. That is, a PCR reaction was performed using a cDNA obtained by reverse transcribing 25 ng of total RNA extracted from mouse EL-4 cells as a template. PCR is
Using F5 and R3 primers, 94 ° C for 30 seconds, 50 ° C
This was performed 33 times with the conditions of 2 minutes and 72 ° C. for 45 seconds as one cycle.

Forward primer F5 primer: 5'-GCNGCNTGGGGNAT-3 '(SEQ ID NO: 26: nucleotide sequence deduced from the 65th to 69th amino acid sequence of the amino acid sequence described in SEQ ID NO: 1) In the sequence, N is A , C, G or T.

Reverse primer R3 primer: 5'-CATGGAGTAGCCTGCATGG-3 '(SEQ ID NO: 2
9) (Sequence complementary to nucleotides 474 to 492 of the nucleotide sequence described in SEQ ID NO: 1) The gene fragment obtained as described above was
It was subcloned into escript II and its nucleotide sequence was determined by the dideoxy method.

As a result, a partial fragment of the gene encoding the mouse tumor antigen peptide was obtained, and its DNA base sequence was represented by SEQ ID NO: 13. The amino acid sequence encoded by the nucleotide sequence is represented by SEQ ID NO: 14, and the RNA sequence is represented by SEQ ID NO: 15. Among the above peptide sequences, the sequence corresponding to the tumor antigen epitope is Phe Ser AlaThr Glu Pro Lys (SEQ ID NO: 3).
0) (called M-2).

Example 3 Chemical Synthesis of Tumor Antigen Peptide The present inventor used a peptide synthesizer (Model 433A) of Applied Biosystems Inc. to synthesize various peptides containing the sequence of the M-1 peptide by the Fastmoc method. Synthesized. The synthesized peptide was Ala10 (Glu Pro Phe Ser Ala Thr Asp Pro L
ys Ala), MAP10 using MAP resin (Ala10
Gly20 (Ala Glu Pro Phe S)
er Ala Thr Asp Pro Lys Ala Ile Gly His Arg Asn Tyr
His Ala Gly) and MAP20 (Gly20 bound to MAP resin in four branches). Further, based on the results obtained in Example 2, a tumor-specific antigen peptide expressed in mouse EL-4 cells was synthesized in the same manner as described above.

Synthesized peptides: M-2: Phe Ser Ala Thr Glu Pro Lys (SEQ ID NO: 30) m-Ala9 (Pro Phe Ser Ala Thr Glu Pro Lys Ala) (SEQ ID NO: 31), m-Ala10 (Glu Pro Phe Ser Ala Thr Glu Pro Lys Ala
) (SEQ ID NO: 32), m-MAP10 (m-Ala10 linked to MAP resin in eight branches), m-Gly20 (Ala Glu Pro Phe Ser Ala Thr Glu Pro Lys A)
la Ile Gly His Arg AsnTyr His Ala Gly) (SEQ ID NO:
33), m-MAP20 (m-Gly20 bonded to MAP resin in four branches)

Example 4 Antitumor Agent Containing Tumor Antigen Peptide (1) Antitumor Effect in Mice
Experiments of subcutaneous and oral (enteric microcapsules or enteric capsules) administration confirmed the antitumor activity and safety of these peptides. Gastric cancer cell line MKN45 (1
× 10 ⁷ ) were transplanted to 4-week-old nude mice and bred for 1 week. In addition, four mice were used in each of the peptide administration group and the control group.

Next, 200 mice were administered to the mice to which the peptide was administered.
Peptide (Ala10) was administered subcutaneously on the back at a dose of μg / 100 g / week. This operation was performed three times at two-week intervals, and the survival status of the mice and the presence or absence of tumor shrinkage were observed. As a result, tumor shrinkage was observed in the mice in the peptide administration group 21 days after tumor implantation, and 4 out of 4 mice survived 28 days after tumor administration. No decrease in body weight was observed in the mice to which the peptide of the present invention was administered. Therefore, it was confirmed that the peptide of the present invention was effective and safe as an antitumor agent.

[0197] Detection embodiment of cancers as Example 5 scintillation indices were administered to tumor-bearing mice KTC-3 antibody ¹³¹ I of the present invention is labeled, the presence of cancer from the distribution of ¹³¹ I Position detected. Five days after the tumor cells TUC-1 (Table 1) were transplanted into the mice, ¹³¹ I-labeled KTC-3 antibody was administered to the mice. After transplantation, ¹³¹ I was detected by a scintillation counter.

As a result, the antibodies of the present invention accumulated in cancer tissues (FIG. 17). In FIG. 17, the upper small scintigram is for the thyroid gland, and the lower large scintigram is for the cancer tissue. Therefore, it was shown that cancer can be detected using the antibody of the present invention and a radioisotope ( ¹³¹ I).

Example 6 Detection of Expression of SSY Tumor Antigen (c ″ Subunit) and c Subunit and a Subunit in Various Tumor Cells The V-ATPase c subunit and a subunit are highly expressed in tumors No consideration has been given as to whether or not this is the case. Therefore, the present inventors examined the expression of the SSY tumor antigen, c subunit and a subunit in various tumor tissues (removed surgical specimens) by RT-PCR, and detected the genes encoding the respective proteins. RT-PCR, TOYO
This was performed using the RT-PCR high kit from BO according to the instruction manual. That is, a PCR reaction was performed using a cDNA obtained by reverse transcribing 25 ng of total RNA extracted from various tumor tissues as a template. The PCR was performed 33 times with the conditions of 94 ° C. for 30 seconds, 56 ° C. for 30 seconds and 72 ° C. for 1 minute as one cycle. The primers used for PCR are as follows.

(1) Primer for SSY tumor antigen gene 1 Forward primer F2 primer (forward primer 2): 5′-TGTGAGGCTGTGGC
CATCTA-3 ′ (SEQ ID NO: 23) (SEQ ID NO: 1)
F3 primer (forward primer 3): 5'-AGACTGCGGGACG-
3 '(SEQ ID NO: 24) (corresponding to the first to 13th sequences of the nucleotide sequence described in SEQ ID NO: 1) 2 Reverse primer R1 primer (reverse primer 1): 5'-CCCCACCCACACAT
ATCATC-3 ′ (SEQ ID NO: 27) (SEQ ID NO: 1)
R3 primer (reverse primer 3): 5'-CATGGAGTAGCCTG
CATGG-3 ′ (SEQ ID NO: 29) (SEQ ID NO: 4
(Sequence complementary to the 74th to 492nd sequence)

(2) Primer for c subunit gene 1 Forward primer Fc primer (forward primer c): 5'-GGCCGCCATGGTCT
TCA-3 ′ (SEQ ID NO: 38) (No. 288 of the nucleotide sequence of SEQ ID NO: 7)
2) Reverse primer Rc primer (reverse primer c): 5'-TGAGGAGGGGTGGT
CTTTAC-3 ′ (SEQ ID NO: 39) (SEQ ID NO: 7
(A sequence complementary to the 31st to 750th sequence)

(3) Primer for a subunit gene 1 Forward primer Fa primer (forward primer a): 5'-CCACCATGGGGGAG
CT-3 ′ (SEQ ID NO: 40) (No. 164 of the nucleotide sequence described in SEQ ID NO: 10)
2) Reverse primer Ra primer (reverse primer a): 5'-CAAGTCTGGATCCG
CCATC-3 ′ (SEQ ID NO: 41) (5th nucleotide sequence of SEQ ID NO: 10)
(A sequence complementary to the 91st to 609th sequences)

(4) Size of Amplified Fragment The size of the DNA fragment amplified by these primers is, for example, 349 bp when using F2 primer and R1 primer, and 463 bp when using Fc primer and Rc primer. And 446 bp when using Fa and Ra primers. As a positive control, a primer (included in the kit) for a housekeeping gene (glyceraldehyde triphosphate dehydrogenase (GAPDH)) was used. The size of the DNA fragment amplified by these primers is 450 bp. It was confirmed that no band appeared when the reverse transcription reaction was not performed (-RT). After the PCR reaction, DNA fragments were confirmed by agarose gel electrophoresis. The size marker is a 100 bp ladder (Pharmacia).

(5) Results As a result, thyroid cancer, sarcoma (rhabdomyosarcoma,
Strong expression of SSY tumor antigen was detected in leukemias and malignant lymphomas (including osteosarcoma species) (FIG. 18). In addition, the c-subunit and a-subunit of V-ATPase were also relatively highly expressed in tumors (breast cancer (adenocarcinoma)) (FIG. 19). However, in FIG. 18, in which slight or almost no detection was detected in the normal tissue corresponding to each organ of the same patient used as a control, the content of the sample in each lane is shown in Table 6, and FIG. 19 is shown in Table 7 for FIG.

[0205]

[Table 6]

[0206]

[Table 7]

Example 7 Antitumor Agent Containing DNA Vaccine (Gene Therapeutic Agent) In this example, the antitumor effect and safety when DNA was directly intramuscularly injected into tumor-bearing mice were examined. First, stomach cancer ce
MKN45 (1 × 10 ⁷ cells) as an ll line was transplanted into 4-week-old nude mice and bred for 1 week. Two mice were used for each of the plasmid DNA administration group and the control group.

On the other hand, Gly20: Ala Glu Pro Phe Ser Ala T
hr Asp Pro Lys Ala Ile Gly His Arg Asn Tyr His A
DNA encoding la Gly was amplified by PCR. The amplified fragment was introduced into a pCMX plasmid having a CMV-IE promoter, purified using a QIAGEN plasmid purification kit, and then dissolved in physiological saline. Next, the plasmid DNA
For mice grouped as a treatment group, 100 μg / 100 g /
Plasmid DNA was injected intramuscularly into the thighs of mice at weekly doses. This operation was performed three times at two-week intervals, and the survival status of the mice and the presence or absence of tumor shrinkage were observed.

As a result, 21 days after tumor implantation, plasmid DN
Tumor shrinkage was observed in the mice in the A administration group, and two out of two mice survived 28 days after tumor administration. In addition, no decrease in body weight was observed in the mice to which the plasmid DNA of the present invention was administered. Therefore, it was confirmed that the plasmid DNA of the present invention was effective and safe as an antitumor agent.

Example 8 Antitumor Effect of Antisense Oligomers The present inventors examined the effects on cell proliferation of TPC1 cells, MKN45 cells and human normal keratinocytes using S-oligomers (phosphorotioate type oligomers). . (1) Preparation of cell suspension Tumor cells (TPC1 cells, MKN45 cells) were trypsinized, suspended in 5 ml of RPMI-1640 medium, and centrifuged at 4 ° C. at 1,000 rpm for 5 minutes. Thereafter, the supernatant was removed by suction and suspended in 5 ml of medium. After passing the suspension through the mesh, the number of cells was counted. That is, 3 samples (500 μl / well) as a stock solution were spread on a 24-well plate, and counted using a cell counter. The dilution was adjusted in a centrifuge tube to 10 × 10 ⁴ cells / ml in the medium. 3 samples (500 μl / well) as cell suspension for 24
Drilled in a hole plate.

(2) Preparation of Antisense Oligomers As S-oligomers, sense (MS) and antisense (MA) for the translation initiation region of the SSY tumor antigen protein, and sense (ES) and antisense for the epitope region of the KTC3 antibody were used. Four types of sense (EA) were synthesized. Their sequences are as follows:

MS: 5'-GCCATGACGGGGCTA-3 '(SEQ ID NO: 42) MA: 5'-TAGCCCCGTCATGGC-3' (SEQ ID NO: 43) ES: 5'-AGTGCCACAGACCCC-3 '(SEQ ID NO: 44) EA: 5'- GGGGTCTGTGGCACT-3 '(SEQ ID NO: 45) These S-oligomers were added to a final concentration of 10 μM in the culture of each cell, and 5% CO ₂ at 37 ° C. for 3 days.
-Incubate in incubator and count cells.

(3) Results 1. Antisense oligomer (EA) in the epitope region
While the proliferation of TPC1 cells and MKN45 cells is significantly inhibited, the proliferation of normal keratinocytes is only slightly inhibited. 2 Sense oligomer (ES) in the same region as the epitope region It was also found that antisense oligomer (MA) in the 3 translation initiation region had the same effect as EA, but its effect was small. This indicates that the antisense oligomer against the SSY tumor antigen specifically suppresses the growth of tumor cells, that is, is useful as an antitumor therapeutic agent.

[Example 9] Tumoricidal effect of anti-c subunit antibody The method for producing an antibody against the c subunit was basically the same as in Nezu
(NezuJ et al., J. Biochem. 122: 21
2-219, 1992). That is, extracellular proteins of tumor cells were selected from c subunits to be used as immunogens (antigens). As a candidate extramembrane protein, a partial sequence of the amino acid sequence of the mouse c subunit (16 KDa) (the amino acid sequence described in SEQ ID NO: 5) was used. That is, Kyte and Doolittle (Kyte JS et al., J. Mol. Biol.) Have a window size of 11 amino acids in the amino acid sequence of the c subunit.
157: 105-132, 1982) and analyzed using the Met Ala Asp Ile Lys Asn Asn Pro Glu Tyr Ser.
Ser Phe Phe Gly Val (1st to 16th sequence of the amino acid sequence described in SEQ ID NO: 5) was used as a candidate.

The above-mentioned candidate peptide was synthesized by a peptide synthesizer, and bound to hemocyanin using glutaraldehyde to obtain an antigen. Rabbits were immunized with the antigen prepared as described above. The antigen dose per rabbit is 0.5 mg when no adjuvant is used, and 100 μg when adjuvant is used. As an adjuvant, carriers such as Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) were used. Administration sites were intradermal and subcutaneous. In addition, immunization was performed four times at two-week intervals. The antibody titer was measured 6 to 60 days after the last immunization day, and blood was collected on the day showing the maximum antibody titer to obtain an antiserum. The method of Goyard et al. (Goyard S et al.,
Biochemistry 28: 1964-1967, 1989). The antibody titer was measured by an enzyme immunoassay (ELISA; enzyme-linked immunosorbent assay), a radioimmunoassay (RIA; radioimmuno assay), or the like.

The anti-c peptide-specific antibody prepared as described above was used in the following antitumor test. In addition, mouse erythroleukemia cells (EL-4) were used as targets. In addition, spleen cells of C57BL / 6 mouse were used as a normal cell control group. Each cell was grown in vitro until the density reached an extreme value. Thereafter, four kinds of anti-tumor antibodies including an anti-c subunit antibody were prepared and serially diluted. Normal rabbit serum was used as an antibody control. The antibody solution at each dilution step was added to the tumor cell culture supernatant and cultured for another 3 days. After culture, DNA fragmentation was determined, and apoptosis-positive cells were calculated.

An outline of the procedure for determining apoptosis by FACS is as follows. First, sample cells were prepared from a culture solution, and fixed with 70% ethanol for 1 to 2 hours. Then 0.125%
Treated with RNase at a concentration of 20 min at 37 ° C and centrifuged (1
(000 rpm, 5 minutes). Sample Pro
The cells were stained with pidium Iodide (final concentration: 10 mg / ml), analyzed with a flow cytometer, and apoptosis positive cells (DNA fragmented cells) were counted.

As a result, apoptosis was selectively induced in tumor cells by the anti-c subunit antibody (FIG. 20).
In FIG. 20, anti-c subunit antibody (4-fold dilution) was added.
The numbers of apoptotic cells counted by FACS after 2 hours (right panel) and 72 hours after addition of normal rabbit serum as a control (left panel) are shown.

Example 10 Antitumor Effect of Various Proton Pump Inhibitors In this example, cell lines derived from Chinese hamster ovarian cancer (Chinese Hamster Ovary Cells: CHO Cells), PC
The antitumor effects of various proton pump inhibitors were examined using cell lines derived from various cancers such as -12, TPC-1, MKN45 and EL-4.

The cell proliferation assay followed the following procedure. (1) Preparation of Cell Suspension After trypsinization of tumor cells, they were suspended in 5 ml of RPMI1640 medium and centrifuged at 1,000 rpm for 5 minutes at 4 ° C. Thereafter, the supernatant was removed by suction and suspended in 5 ml of medium. After passing the suspension through the mesh, the number of cells was counted. That is, 3 samples (500 μl / well) as a stock solution were spread on a 24-well plate, and counted using a cell counter. The dilution was adjusted in a centrifuge tube to 10 × 10 ⁴ cells / ml in the medium. 3 as cell suspension
The sample (500 μl / well) was spread on a 24-well plate.

(2) Preparation of Drug The following drugs were used as drugs. (2-1) V-ATPase inhibitor EDTA, bafilomycin A1, bafilomycin B1, EGTA, concanamycin A, concanamycin B, O-methylconcanamycin, N-ethylmaleimide, N, N'-dicyclohexylcarbodiimide , Loikanicidin, 7-chloro-4-nitrobenz (o) -2-oxa-1,3-diazole, suramin, verapamil, (2Z, 4E) -5- (5,6-dichloro-2-indolyl) -2- Methoxy-N
-(1,2,2,6,6-pentamethylpiperidin-4-yl) -2,4-pentadienamide, destraxin B, destraxin E, OST-766, 5- (5,6-dichloro-2 -Indolyl) -2-methoxy-2,4-pentadienamide, NO ₃ , 3'-O- (4-benzoyl) benzoyladenosine 5'-triphosphate, hygrolizine, bisphosphonate tiludronate, diethylpyrocarbonate, dibromoacetophenone, p -Chloromercuric benzoic acid, quercetin, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, N-bromo Succinimide, benzoyltaxinin K, (2-2) H + / K + -ATPase inhibitor (2-2-1) Benzimidazole derivative, rabeprazole sodium, omeprazole, pantoprazole, reminoprazole, 2-dimethylamino-4,5 -Dihydrothiazolo [4,5: 3,4] pyri [1,2-a] benzimidazole, 2-aryl-4,5-dihydro-1H-thieno [3,2-e] benzimidazole, (2-2-2) Other than benzimidazole derivatives (±)- 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyrido
-2-yl) methyl] sulfinyl] -1H-imidazo (4,5-b) pyridine, 3-amino-5-methyl-2- (2-methyl-3-thienyl) imidazo
[1,2-a] thieno [3,2-c] pyridine, 1-aryl-3-substituted pyrrolo [3,2-c] quinoline, 3-butyryl-8-methoxy-4-[(2-thiophenyl) Amino] quinoline, 2-[(3,5-dimethyl-4-methoxypyridyl) alkyl] -benzothiazolidine, 3-butyryl-4- [R-1-methylbenzylamino] -8-ethoxy
-1,7-naphthyridine, pentagalloylglucose (2-3) H + / Cl-symporter prodigiosin, metacycloprodigiosin, prodigiosin 25-C, cycloprodigiosin, cycloprodigiosin hydrochloride, bepridil (1 -Isobutoxy-2-pyrrolidino-3- [N-benzylanilino] propane) manganese ・ tetraphenylporphine, thallium chloride (TlCl ₃ )

The preparation of the medicament is carried out, for example, by using bafilomycin A1.
In case (1), 1 mM bafilomycin A1 was suspended in 5 μl of DMSO, and a 4-fold dilution series was prepared thereafter and used as a positive control in the experiment. Also, sodium rabeprazole (Sodium Rabepr
azole) dissolved in 10% FCS-RPMI 1640 (4mg / ml)
0.02 to 2 mg / ml was added to TPC-1 cells.

(3) Cell Culture The cell suspension was spread on a 24-well plate at 500 μl / well, and 500 μl / well of a drug-containing medium was added to the 24-well plate. The cells were incubated at 37 ° C. for 3 days in a 5% CO ₂ -incubator, and the number of cells was counted. (4) Results The above drug was confirmed to have a tumor cell growth inhibitory effect, and was shown to be useful as an antitumor agent (FIGS. 21 to 21).
29, Figures 31-36). The chemical structural formulas of the proton pump inhibitors shown in FIGS. 21 to 29 are shown in FIGS. Fig. 30
A to C show V-ATPase inhibitors, and FIG. 30D shows prodigiosins (H + / Cl-symporters) among H + / K + -ATPase inhibitors.

Example 11 Antitumor Activity of Leukocyte of the Present Invention Using DC cells dendritic cells pulsed with a tumor antigen peptide, the peptide-specific immunity was induced to test cancer cell destruction activity. That is, one of the following two methods was selected as a method for separating DC. 1. From DC precursor cells in peripheral blood, Ficoll, Percoll, M
The DC fraction obtained by the continuous density gradient separation method using etrizamide was cultured for 24-48 hours, and separated without using cytokines by collecting dish adherent cells.
Overlaid on 15% Metrizamide, centrifuged at 2000 rpm for 10 minutes, and pulsed with various tumor antigen peptides into the DC fraction obtained,
After further culturing for 12-18 hours, the cells were overlaid on 14% Metrizamide and centrifuged at 2000 rpm for 10 minutes to recover mature DC.

In the presence of IL-4 (400 U / ml) and TNFα (100 U / ml)
DCs were induced by culturing for 5-7 days, tumor antigen peptides were added to a final concentration of 20-30 μM, and overnight pulse was performed to collect 1 × 10 ⁵ to 1 × 10 ⁶ DCs. The tumor peptide used was Ser Asn Met Ala Glu Pro Phe Ser Ala Th
r Glu Pro Lys Ala Ile Gly His Arg Asn Tyr (43-62 in SEQ ID NO: 14).

The preparation of the tumor-bearing mice was performed as follows.
That is, 1 × 10 ⁷ EL-4 cells were subcutaneously transplanted into 4-week-old nude mice and bred for 1 week, and the DC cells were intravenously injected into the mice, and thereafter, the presence or absence of tumor shrinkage was observed. Two mice were used for the DC administration group and two control groups.

As a result, tumor shrinkage (10% and 15% reduction ratios respectively in comparison with the control group) was observed in the DC cell-administered mice 21 days after tumor implantation, and 28 days after tumor inoculation. However, the mice survived, and no abnormalities such as weight loss were observed in the DC-administered mice. From the above, the present invention
DC was confirmed to be effective and safe as an antitumor agent.

[0228]

According to the present invention, a tumor-specific antigen peptide,
Antibodies to the peptide and pharmaceutical compositions comprising the peptide or antibody are provided. Since the peptides and antibodies of the present invention have an antitumor effect, they are useful for treating and preventing cancer, and are also useful for diagnosing cancer and the like.

[0229]

[Sequence List] SEQUENCE LISTING <110> Satoh, Nobuo Suzuki, Nobutaka Yamaguchi, Masaaki <120> Tumor-specific antigenic peptides <130> P00-0231 <140> <141> <160> 52 <170> PatentIn Ver. 2.0 <210> 1 <211> 986 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (82) .. (696) <400> 1 agactgcggg acggacggtg gacgctggga cgcgtttgta gctccggccc cgccgttccg 60 acccccgccg ccgtcgggg c atg acg ggg cta gca ctg ctc tac tcc ggg 111 Met Thr Gly Leu Ala Leu Lec g gt gt gt gt gt gt gt gt gt gt gt gt g gga gtc tgc tac 159 Val Phe Val Ala Phe Trp Ala Cys Ala Leu Ala Val Gly Val Cys Tyr 15 20 25 acc att ttt gat ttg ggc ttc cgc ttt gat gtg gca tgg ttc ctg acg 207 Thr Ile Phe Asp Leu Gly Phe Asp Val Ala Trp Phe Leu Thr 30 35 40 gag act tcg ccc ttc atg tgg tcc aac ctg ggc att ggc cta gct atc 255 Glu Thr Ser Pro Phe Met Trp Ser Asn Leu Gly Ile Gly Leu Ala Ile 45 50 55 tcc ctg tct gtg gtt ggg gca gcc tgg ggc atc tat att acc ggc tcc 303 Ser Leu Ser Val Val Gly Ala Ala Trp Gly Ile Tyr Ile Thr Gly Ser 60 65 70 tcc atc att ggt gga gga gtg aag gcc ccc agg atc aag acc aag aac 351 Ser Ile Ile Gly Gly Gly Val Lys Ala Pro Arg Ile Lys Thr Lys Asn 75 80 85 90 ctg gtc agc atc atc ttc tgt gag gct gtg gcc atc tac ggc atc atc 399 Leu Val Ser Ile Ile Phe Cys Glu Ala Val Ala Ile Tyr Gly Ile Ile 95 100 105 atg gca att gtc att agc aac atg gct gag cct ttc agt gcc aca gac 447 Met Ala Ile Val Ile Ser Asn Met Ala Glu Pro Phe Ser Ala Thr Asp 110 115 120 ccc aag gcc atc ggc cat cgg aac tac cat gca ggc tac tcc atg ttt 495 Pro Lys Ala Ile Gly His Arg Asn Tyr His Ala Gly Tyr Ser Met Phe 125 130 135 ggg gct ggc ctc acc gta ggc ctg tct aac ctc ttc tgt gga gtc tg Gly Ala Gly Leu Thr Val Gly Leu Ser Asn Leu Phe Cys Gly Val Cys 140 145 150 gtg ggc atc gtg ggc agt ggg gct gcc ctg gcc gat gct cag aac ccc 591 Val Gly Ile Val Gly Ser Gly Ala Ala Leu Ala Asp Ala Gln Asn Pro 155 160 165 170 agc ctc ttt gta aag att ctc atc gtg gag atc ttt ggc agc gcc att 639 Ser Leu Phe Val Lys Ile Leu Ile Val Glu Ile Phe Gly Ser Ala Ile 175 180 185 ggc ctc ttt ggg gtc atc att ctt cag acc tcc aga gtg aag 687 Gly Leu Phe Gly Val Ile Val Ala Ile Leu Gln Thr Ser Arg Val Lys 190 195 200 atg ggt gac tagatgatat gtgtgggtgg ggccgtgcct cacttttatt 736 Met Gly Asp 205 tattgctggt tt tt tcctggga cagctggagc tgtgtccctt agcctttcag aggcttggtg 796 ttcagggccc tccctgcact cccctcttgc tgcgtgttga tttggaggca ctgcagtcca 856 ggccgagtcc tcagtgcggg gagcaggctg ctgctgctga ctctgtgcag ctgcgcacct 916 gtgtccccca cctccaccct caacccatct tcctagtgtt tgtgaaataa acttggtatt 976 tgtctgggtc 986 <210> 2 <211> 205 <212> PRT <213> Homo sapiens <400> 2 Met Thr Gly Leu Ala Leu Leu Tyr Ser Gly Val Phe Val Ala Phe Trp 1 5 10 15 Ala Cys Ala Leu Ala Val Gly Val Cys Tyr Thr Ile Phe Asp Leu Gly 20 25 30 Phe Arg Phe Asp Val Ala Trp Phe Leu Thr Glu Thr Ser Pro Phe Met 35 40 45 Trp Ser Asn Leu Gly Ile Gly Leu Ala Ile Ser Leu Ser Val Val Gly 50 55 60 Ala Ala Trp Gly Ile Tyr Ile Thr Gly Ser Ser Ile Ile Gly Gly Gly 65 70 75 80 Val Lys Ala Pro Arg Ile Lys Thr Lys Asn Leu Val Ser Ile Ile Phe 85 90 95 Cys Glu Ala Val Ala Ile Tyr Gly Ile Ile Met Ala Ile Val Ile Ser 100 105 110 Asn Met Ala Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala Ile Gly His 115 120 125 Arg Asn Tyr His Ala Gly Tyr Ser Met Phe Gly Ala Gly Leu Thr Val 130 135 140 Gly Leu Ser Asn Leu Phe Cys Gly Val Cys Val Gly Ile Val Gly Ser 145 150 155 160 Gly Ala Ala Leu Ala Asp Ala Gln Asn Pro Ser Leu Phe Val Lys Ile 165 170 175 Leu Ile Val Glu Ile Phe Gly Ser Ala Ile Gly Leu Phe Gly Val Ile 180 185 190 Val Ala Ile Leu Gln Thr Ser Arg Val Lys Met Gly Asp 195 200 205 <210> 3 <211> 986 <212> RNA <213> Homo sapiens <400> 3 agacugcggg acggacggug gacgcuggga cgcguuugua gcuccggccc cgccguuccg 60 acccccgccg ccgucgccgc caugacgggg cuagcacugc ucuacuccgg ggucuucgug 120 gccuucuggg ccugcgcgcu ggccguggga gucugcuaca ccauuuuuga uuugggcuuc 180 cgcuuugaug uggcaugguu ccugacggag acuucgcccu ucaugugguc caaccugggc 240 auuggccuag cuaucucccu gucugugguu ggggcagccu ggggcaucua uauuaccggc 300 uccuccauca uugguggagg agugaaggcc cccaggauca agaccaagaa ccuggucagc 360 aucaucuucu gugaggcugu ggccaucuac ggcaucauca uggcaauugu cauuagcaac 420 auggcugagc cuuucagugc cacagacccc aaggccaucg gccaucggaa cuaccaugca 480 ggcuacucca uguuuggggc uggccucacc guaggccugu cuaaccucuu cuguggaguc 540 ugcgugggca ucgugggcag uggggcugcc cuggccgaug cucagaaccc cagccucuuu 600 guaaagauuc ucaucgugga gaucuuuggc agcgccauug gccucuuugg ggucaucguc 660 gcaauucuuc agaccuccag agugaagaug ggugacuaga ugauaugugu ggguggggcc 720 gugccucacu uuuauuuauu gcugguuuuc cugggacagc uggagcugug ucccuuagcc 780 uuucagaggc uugguguuca gggcccuccc ugcacucccc ucuugcugcg uguugauuug 840 gaggcacugc ag uccaggcc gaguccucag ugcggggagc aggcugcugc ugcugacucu 900 gugcagcugc gcaccugugu cccccaccuc cacccucaac ccaucuuccu aguguuugug 960 aaauaaacuu gguauuuguc uggguc 986 <210> 4 <211> 1153 <212> DNA <213> Mus musculus <220> <221> CDS <222> (148) .. (612) <400> 4 aattccggta tttagagcgc agcgcctgac tggcggggtc gctttcccat ccgctgcctc 60 gacgatcttc gcttgcctcc tcgctcgctg tcccgttgtc ctagcccgcc gccgcccgct 120 gagcttgtct tttccctgct tgcagac atg gct gac atc aag aac aac ccc gaa 174 Met Ala Asp Ile Lys Asn Asn Pro Glu 1 5 tat tct tcg ttt ttc ggt gtc atg ggc gcc tcg tcc gcc atg gtc ttc 222 Tyr Ser Ser Phe Phe Gly Val Met Gly Ala Ser Ser Ala Met Val Phe 10 15 20 25 agc gcc atg gga gcc gcc tat ggc aca gcc aag agc ggc act ggc atc 270 Ser Ala Met Gly Ala Ala Tyr Gly Thr Ala Lys Ser Gly Thr Gly Ile 30 35 40 gca gcc atg tca gtc atg agg cca gag ctg atc atg aag tcc atc atc 318 Ala Ala Met Ser Val Met Arg Pro Glu Leu Ile Met Lys Ser Ile Ile 45 50 55 cca gtg gtt atg gct ggg atc atc gcc atc tac ggc ctg gtg gtg gca 366 Pro Val Val Met Ala Gly Ile Ile Ala Ile Tyr Gly Leu Val Val Ala 60 65 70 gta ctt atc gct aac tcc ctg act gat ggc atc acc ctc tac agg agt 414 Val Leu Ile Ala Asn Ser Leu Thr Asp Gly Ile Thr Leu Tyr Arg Ser 75 80 85 ttt ctt caa ctg ggt gct ggc ctg agt gtg ggg ctg agt ggc ctg gct 462 Phe Leu Gln Leu Gly Ala Gly Leu Ser Val Gly Leu Ser Gly Leu Ala 90 95 100 105 gct ggc ttt gcc att ggc atc gtc gga gat gct ggt gtt cgg ggc act 510 Ala Gly Phe Ala Gly Ile Val Gly Asp Ala Gly Val Arg Gly Thr 110 115 120 gcc cag cag cct cga ctg ttc gtg ggc atg atc ctg atc ctc atc ttt 558 Ala Gln Gln Pro Arg Leu Phe Val Gly Met Ile Leu Ile Leu Ile Phe 125 130 135 gcg gag gtg ctt ggc ctc tac ggt ctc atc gtg gcc cta atc ctc tcc 606 Ala Glu Val Leu Gly Leu Tyr Gly Leu Ile Val Ala Leu Ile Leu Ser 140 145 150 aca aag tagtcctttt ccaccatcag tcacag gag ccat ccat gag tt cacgggcagc tgcccctcag tagttggtct 722 tgtaaatgtg cagtgtccta gtgcccattg tctgtcgccc ccgccttgcc cctactgcac 782 cgtgctgtgg acatacgggc ccactcatct cccacccagg cccctgacca gtgacgaagt 842 cagcctctgg ttaccccacc catcgcccta gagtgctcct gtgtataaga atgaactaga 902 gttgtcattt ttctcttcac tggatgttta tttataaaag atttgaccta ttcatgcgtc 962 tgtggagcag ctcctgtctc ccaact atat agtaatcatt agtagactgt tgccttgtgg 1022 ggttcctgtt gctgagactc cttggatgga gccacccttc cccctgccct gaacagccag 1082 ggtggaggat agagtgctac tgctcctcag agctgg cagctgtgtc caataaaagt114g <210> 5 <211> 155 <212> PRT <213> Mus musculus <400> 5 Met Ala Asp Ile Lys Asn Asn Pro Glu Tyr Ser Ser Phe Phe Gly Val 1 5 10 15 Met Gly Ala Ser Ser Ala Met Val Phe Ser Ala Met Gly Ala Ala Tla 20 25 30 Gly Thr Ala Lys Ser Gly Thr Gly Ile Ala Ala Met Ser Val Met Arg 35 40 45 Pro Glu Leu Ile Met Lys Ser Ile Ile Pro Val Val Met Ala Gly Ile 50 55 60 Ile Ala Ile Tyr Gly Leu Val Val Ala Val Leu Ile Ala Asn Ser Leu 65 70 75 80 Thr Asp Gly Ile Thr Leu Tyr Arg Ser Phe Leu Gln Leu Gly Ala Gly 85 90 95 Leu Ser Val Gly Leu Ser Gly Leu Ala Ala Gly Phe Ala Ile Gly Ile 100 105 110 Val Gly Asp Ala Gly Val Arg Gly Thr Ala Gln Gln Pro Arg Leu Phe 115 120 125 Val Gly Met Ile Leu Ile Leu Ile Phe Ala Glu Val Leu Gly Leu Tyr 130 135 140 Gly Leu Ile Val Ala Leu Ile Leu Ser Thr Lys 145 150 155 <210> 6 <211> 1153 <212> RNA <213> Mus musculus <400> 6 aauuccggua uuuagagcgc agcgccugac uggcgggguc gcuuucccau ccgcugccuc 60 gacgaucuuc gcuugccucc ucgcucgcug ucccguuguc cuagcccgcc gccgcccgcu 120 gagcuugucu uuucccugcu ugcagacaug gcugacauca agaacaaccc cgaauauucu 180 ucguuuuucg gugucauggg cgccucgucc gccauggucu ucagcgccau gggagccgcc 240 uauggcacag ccaagagcgg cacuggcauc gcagccaugu cagucaugag gccagagcug 300 aucaugaagu ccaucauccc agugguuaug gcugggauca ucgccaucua cggccuggug 360 guggcaguac uuaucgcuaa cucccugacu gauggcauca cccucuacag gaguuuucuu 420 caacugggug cuggccugag uguggggcug aguggccugg cugcuggcuu ugccauuggc 480 aucgucggag augcuggugu ucggggcacu gcccagcagc cucgacuguu cgugggcaug 540 auccugaucc ucaucuuugc ggaggugcuu ggccucuacg gucucaucgu ggcccuaauc 600 cucuccacaa aguaguccuu uuccaccauc agucacagga uaggauguaa agaucacccc 660 uccucauucc agaacgaaca gccugacaca ugcacgggca gcugccccuc aguaguuggu 720 cuuguaaaug ugcagugucc uagugcccau ugucugucgc ccccgccuug ccccuacugc 780 accgugcugu ggacauacgg gcccacucau cucccaccca ggccccugac cagugacgaa 840 gucagccucu gg uuacccca cccaucgccc uagagugcuc cuguguauaa gaaugaacua 900 gaguugucau uuuucucuuc acuggauguu uauuuauaaa agauuugacc uauucaugcg 960 ucuguggagc agcuccuguc ucccaacuau auaguaauca uuaguagacu guugccuugu 1020 gggguuccug uugcugagac uccuuggaug gagccacccu ucccccugcc cugaacagcc 1080 aggguggagg auagagugcu acugcuccuc agagcuggcu cccagcugug uccaauaaaa 1140 guucucggau gug 1153 <210> 7 <211> 1160 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (229) .. (693) <400> 7 gcgtctcccc cacggtgcga agtgggtacg gctcgcaggg gcggggccag gtcatgtgac 60 gcggccgcgc cgcattttgt tctgcggtgc tggtatttag agcgcacgct gacgggccgg 120 atcgccttcg ccgccgcccg cccgcaaacc ttcgtgcccg gcccgtcctc gcccccgcct 180 ccgccaccgc ctcggcccgc agagcttgcc ccctccccac ccgcagac atg tcc gag 237 Met Ser Glu 1 tcc aag agc ggc ccc gag tat gct tcg ttt ttc gcc gtc atg ggc gcc 285 Ser Lys Ser Gly Pro Glu Tyr Ala Ser Phe Phe Ala Val Met Gly Ala 5 10 15 tcg gcc gcc atg gtc ttc agc gcc ctg ggc gct gcc tat ggc aca gcc 333 Ser Ala Ala Met Val Phe Ser Ala Le Gly Ala Ala Tyr Gly Thr Ala 20 25 30 35 aag agc ggt acc ggc att gcg gcc atg tct gtc atg cgg ccg gag cag 381 Lys Ser Gly Thr Gly Ile Ala Ala Met Ser Val Met Arg Pro Glu Gln 40 45 50 atc atg aag tcc atc atc cca gtg gtc atg gct ggc atc atc gcc atc 429 Ile Met Lys Ser Ile Ile Pro Val Val Met Ala Gly Ile Ile Ala Ile 55 60 65 tac ggc ctg gtg gtg gca gtc ctc atc gcc aac tcc gac gat Tyr Gly Leu Val Val Ala Val Leu Ile Ala Asn Ser Leu Asn Asp Asp 70 75 80 atc agc ctc tac aag agc ttc ctc cag ctg ggc gcc ggc ctg agc gtg 525 Ile Ser Leu Tyr Lys Ser Phe Leu Gln Leu Gly Ala Gly Leu Ser Val 85 90 95 ggc ctg agc ggc ccg gcc gcc gcc atc gtg ggg gac 573 Gly Leu Ser Gly Leu Ala Ala Gly Phe Ala Ile Gly Ile Val Gly Asp 100 105 110 115 gct ggc gtg cgg ggc acc gcc cag cag ccc cga cta ttc gtg ggc atg 621 Ala Gly Val Arg Gly Thr Ala Gln Gln Pro Arg Leu Phe Val Gly Met 120 125 130 atc ctg att ctc atc ttc gcc gag gtg ctc ggc ctc tac ggt ctc atc 669 Ile Leu Ile Leu Ile Phe Ala Glu Val Leu Gly Leu Tyr Gly Leu Ile 135 140 145 gtc g atc ctc tcc aca aag tagaccctct ccgagcccac cagccacaga 723 Val Ala Leu Ile Leu Ser Thr Lys 150 155 atattatgta aagaccaccc ctcctcattc cagaacgaac agcctgacac atacgcagcg 783 ccgcccgccc ccagtagttg gtcttgtaca tgcgcagtat cctagtgccc atcgtctgtt 843 tccccgcctt gcccccgccc gccccgtgcc gtggacatct gggcccactc atcgcccctc 903 caggcccccg gcgccccacc ccctaaagtg ctctagtatg cggatgattt agaattgtca 963 tttctcttta ctggatgttt attat taaag atctcgcctg ttcctgcgtc tgcggagccg 1023 cccttgtctc ccagctatct ataaccttag cttgtgtgtc gccttgtggg ttcctgtttcc 1083 tgagactttt cctggatgga gccgccctca ccgcgc gtcgg gtcgg gtcgg gtcgg gtcgg gtcgg gtcgg gtcgg gtcgg gtcgg gtcgg1gt <210> 8 <211> 155 <212> PRT <213> Homo sapiens <400> 8 Met Ser Glu Ser Lys Ser Gly Pro Glu Tyr Ala Ser Phe Phe Ala Val 1 5 10 15 Met Gly Ala Ser Ala Ala Met Val Phe Ser Ala Leu Gly Ala Ala Tyr 20 25 30 Gly Thr Ala Lys Ser Gly Thr Gly Ile Ala Ala Met Ser Val Met Arg 35 40 45 Pro Glu Gln Ile Met Lys Ser Ile Ile Pro Val Val Met Ala Gly Ile 50 55 60 Ile Ala Ile Tyr Gly Leu Val Val Ala Val Leu Ile Ala Asn Ser Leu 65 70 75 80 Asn Asp Asp Ile Ser Leu Tyr Lys Ser Phe Leu Gln Leu Gly Ala Gly 85 90 95 Leu Ser Val Gly Leu Ser Gly Leu Ala Ala Gly Phe Ala Ile Gly Ile 100 105 110 Val Gly Asp Ala Gly Val Arg Gly Thr Ala Gln Gln Pro Arg Leu Phe 115 120 125 Val Gly Met Ile Leu Ile Leu Ile Phe Ala Glu Val Leu Gly Leu Tyr 130 135 140 Gly Leu Ile Val Ala Leu Ile Leu Ser Thr Lys 145 150 155 <210> 9 <211> 1160 <212> RNA <213> Homo sapiens <400> 9 gcgucucccc cacggugcga aguggguacg gcucgcaggg gcggggccag gucaugugac 60 gcggccgcgc cgcauuuugu ucugcggugc ugguauuuag agcgcacgcu gacgggccgg 120 aucgccuucg ccgccgcccg cccgcaaacc uucgugcccg gcccguccuc gcccccgccu 180 ccgccaccgc cucggcccgc agagcuugcc cccuccccac ccgcagacau guccgagucc 240 aagagcggcc ccgaguaugc uucguuuuuc gccgucaugg gcgccucggc cgccaugguc 300 uucagcgccc ugggcgcugc cuauggcaca gccaagagcg guaccggcau ugcggccaug 360 ucugucaugc ggccggagca gaucaugaag uccaucaucc caguggucau ggcuggcauc 420 aucgccaucu acggccuggu gguggcaguc cucaucgcca acucccugaa ugacgacauc 480 agccucuaca agagcuuccu ccagcugggc gccggccuga gcgugggccu gagcggccug 540 gcagccggcu uugccaucgg caucgugggg gacgcuggcg ugcggggcac cgcccagcag 600 ccccgacuau ucgugggcau gauccugauu cucaucuucg ccgaggugcu cggccucuac 660 ggucucaucg ucgcccucau ccucuccaca aaguagaccc ucuccgagcc caccagccac 720 agaauauuau guaaagacca ccccuccuca uuccagaacg aacagccuga cacauacgca 780 gcgccgcccg cccccaguag uuggucuugu acaugcgcag uauccuagug cccaucgucu 840 guuuccccgc cu ugcccccg cccgccccgu gccguggaca ucugggccca cucaucgccc 900 cuccaggccc ccggcgcccc acccccuaaa gugcucuagu augcggauga uuuagaauug 960 ucauuucucu uuacuggaug uuuauuauua aagaucucgc cuguuccugc gucugcggag 1020 ccgcccuugu cucccagcua ucuauaaccu uagcuugugu gucgccuugu ggguuccugu 1080 ugcugagacu uuuccuggau ggagccgccc ucaccgcgcc cguggcccug cgcggagcug 1140 uguccaauaa aguucuugcu 1160 <210> 10 <211> 3032 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (169) .. (2661) <400> 10 gggcggcgca ggggcggggc tttacggacg caagcacgtc gaagcgctgc tcctggagcc 60 gcggagggtg cgggtttggc tgcggtggtt tctgtggcgg ttgctgtggc ggagtttgga 120 ggttggagag aaatccaggt actcactaga ctggtacctt ctgccacc atg ggg gag 177 Met Gly Glu 1 ctt ttc cgg agt gaa gaa atg aca ctg gcc cag ctt ttt cta cag tca 225 Leu Phe Arg Ser Glu Glu Met Thr Leu Ala Gln Leu Phe Leu Gln Ser 5 10 15 gag gct gct tat tgt tgt gtc agt gaa tta gga gaa ctt gga aag gtt 273 Glu Ala Ala Tyr Cys Cys Val Ser Glu Leu Gly Glu Leu Gly Lys Val 20 25 30 35 cag ttt cgt gac tta aat cca gat gtg aat gtt ttc caa cgg aaa ttt 321 Gln Phe Arg Asp Leu Asn Pro Asp Val Asn Val Phe Gln Arg Lys Phe 40 45 50 gtg aat gaa gtt aga aga tgt gaa atg gat cga aag ctt cga ttt 369 Val Asn Glu Val Arg Arg Cys Glu Glu Met Asp Arg Lys Leu Arg Phe 55 60 65 gtt gag aaa gag ata aga aaa gct aac att ccg att atg gac acc ggt 417 Val Glu Lys Glu Ile Arg Lys Ala Asn Ile Pro Ile Met Asp Thr Gly 70 75 80 gaa aac cca gag gtt ccc ttc ccc cgg gac atg atc gac tta gag gc c 465 Glu Asn Pro Glu Val Pro Phe Pro Arg Asp Met Ile Asp Leu Glu Ala 85 90 95 aat ttt gag aag att gaa aat gaa ctg aag gaa atc aac aca aac cag 513 Asn Phe Glu Lys Ile Glu Asn Glu Leu Lys Glu Ile Asn Thr Asn Gln 100 105 110 115 gaa gct ctg aag aga aac ttc ctg gaa ctg acc gaa tta aaa ttt ata 561 Glu Ala Leu Lys Arg Asn Phe Leu Glu Leu Thr Glu Leu Lys Phe Ile 120 125 130 ctt cgc aaa act cag caa ttt ttt gat gag atg gcg gat cca gac ttg 609 Leu Arg Lys Thr Gln Gln Phe Phe Asp Glu Met Ala Asp Pro Asp Leu 135 140 145 ttg gaa gag tcc tca tcc ctc ttg gag cca agt gag atg gga Gag ggclu Ser Ser Ser Leu Leu Glu Pro Ser Glu Met Gly Arg Gly 150 155 160 act cct tta aga ctt ggc ttc gtg gct ggt gtc att aac cgg gag cgc 705 Thr Pro Leu Arg Leu Gly Phe Val Ala Gly Val Ile Asn Arg Glu Arg 165 170 175 atc cct act ttt gag cgc atg ctt tgg cgg gta tgc cgg gga aat gtg 753 Ile Pro Thr Phe Glu Arg Met Leu Trp Arg Val Cys Arg Gly Asn Val 180 185 190 195 ttc ctg cga cag gct gaa atc gag aac ccc gag g at cct gtg act 801 Phe Leu Arg Gln Ala Glu Ile Glu Asn Pro Leu Glu Asp Pro Val Thr 200 205 210 ggc gac tac gtg cac aag tct gtg ttt atc att ttc ttc caa ggc gat 849 Gly Asp Tyr Val His Lys Ser Val Phe Ile Ile Phe Phe Gln Gly Asp 215 220 225 cag ctg aaa aac aga gtc aag aaa atc tgt gaa ggg ttc cga gcc tca 897 Gln Leu Lys Asn Arg Val Lys Lys Ile Cys Glu Gly Phe Arg Ala Ser 230 235 tct c tat c cct gag aca cca cag gag agg aag gaa atg gct tct 945 Leu Tyr Pro Cys Pro Glu Thr Pro Gln Glu Arg Lys Glu Met Ala Ser 245 250 255 gga gtg aat acc agg att gat gat ctc caa atg gtt ctg aat caa acg 993 Gly Val Asn Thr Arg Ile Asp Asp Leu Gln Met Val Leu Asn Gln Thr 260 265 270 275 gag gat cac cgc cag agg gtt ctg cag gca gct gct aag aac atc cgt 1041 Glu Asp His Arg Gln Arg Val Leu Gln Ala Ala Ala Lys Asn Ile Arg 280 285 290 gtc tgg ttc atc aaa gtg cgg aag atg aag gcc atc tat cac acc ctg 1089 Val Trp Phe Ile Lys Val Arg Lys Met Lys Ala Ile Tyr His Thr Leu 295 300 305 aac ctg tgc aac ata gat gtg act aaa tgc ttg att gca gag gtc 1137 Asn Leu Cys Asn Ile Asp Val Thr Gln Lys Cys Leu Ile Ala Glu Val 310 315 320 tgg tgc cct gtc acc gac ctt gac tcc atc cag ttt gca ctc aga agg 1185 Trp Cys Pro Val Asp Leu Asp Ser Ile Gln Phe Ala Leu Arg Arg 325 330 335 ggc acg gaa cac agt ggt tcc act gta cct tcc att ttg aac agg ag 1233 Gly Thr Glu His Ser Gly Ser Thr Val Pro Ser Ile Leu Asn Arg Met 340 345 350 355 cag aca aac cag act ccc cca acc tat aac aaa acc aac aag ttt acc 1281 Gln Thr Asn Gln Thr Pro Pro Thr Tyr Asn Lys Thr Asn Lys Phe Thr 360 365 370 tat ggc ttt cag aac ata gta gat gct tat gga att gga act tac cga 1329 Tyr Gly Phe Gln Asn Ile Val Asp Ala Tyr Gly Ile Gly Thr Tyr Arg 375 380 385 gag ata aat cca gct ccg tat act att atc acg ttc cct ttt cta ttt 1377 Glu Ile Asn Pro Ala Pro Tyr Thr Ile Ile Thr Phe Pro Phe Leu Phe 390 395 400 gct gtg atg ttt gga gac ttc ggt cat ggc att tta atg acc ctt ttt 1425 Ala Val Met Phe Gly Asp Phe Gly His Gly Ile Leu Met Thr Leu Phe 405 410 415 gct gtg tgg atg gta ctg agg gag agc cgg atc ctt tcc cag aag aat 1473 Ala Val Trp Met Val Leu Arg Glu Ser Arg Ile Leu Ser Gln Lys Asn 420 425 430 435 gag aat gag atg ttt agc act gtg ttc agt ggt cga tac att att att tt15 Glu Asn Glu Met Phe Ser Thr Val Phe Ser Gly Arg Tyr Ile Ile Leu 440 445 450 ttg atg ggt gtg ttc tcc atg tac act ggc ctc atc tac aat gat tgc 1569 Leu Met Gly Val Phe Ser Met Tyr Thr Gly Leu Ile Tyr Asn Asp Cys 455 460 465 ttt tcc aag tct ctt aat atc ttt ggg tca tcc tgg agt gta cgg ccg 1617 Phe Ser Lys Ser Leu Asn Ile Phe Gly Ser Ser Trp Ser Val Arg Pro 470 475 480 atg ttt act tat aat tgg act gaa gag acg ctt cgg ggg aac cct gtt 1665 Met Phe Thr Tyr Asn Trp Thr Glu Glu Thr Leu Arg Gly Asn Pro Val 485 490 495 cta cag ctg aac cca gcc ctc cct gga gtg ttt ggt gga cca tac cct 1713 Leu Gln Leu Asn Pro Ala Leu Pro Gly Val Phe Gly Gly Pro Tyr Pro 500 505 510 515 ttt ggc att gat cca att tgg aac att gct acc aat aaa ctg acg ttc 1761 Phe Gly Ile Asp Pro Ile Trp Asn Ile Ala Thr Asn Lys Leu Thr Phe 520 5 25 530 ttg aac tcc ttt aag atg aag atg tct gtt atc ctt ggt atc atc cat 1809 Leu Asn Ser Phe Lys Met Lys Met Ser Val Ile Leu Gly Ile Ile His 535 540 545 atg ctg ttt gga gtc agc ctg agt ctg ttc atc tat ttc aag 1857 Met Leu Phe Gly Val Ser Leu Ser Leu Phe Asn His Ile Tyr Phe Lys 550 555 560 aag ccc ctg aat atc tac ttt gga ttt att cct gaa ata atc ttc atg 1905 Lys Pro Leu Asn Ile Tyr Phe Gly Ile Pro Glu Ile Ile Phe Met 565 570 575 acc tct ttg ttt ggc tat ttg gtt atc ctt att ttt tac aag tgg acg 1953 Thr Ser Leu Phe Gly Tyr Leu Val Ile Leu Ile Phe Tyr Lys Trp Thr 580 585 585 590 595 gcc tat gat gct cat acc tct gag aat gca cca agc ctt ctg atc cat 2001 Ala Tyr Asp Ala His Thr Ser Glu Asn Ala Pro Ser Leu Leu Ile His 600 600 605 610 ttc ata aac atg ttc ctc ttt tcc tac cca gag tct ggt tat tca atg 2049 Phe Ile Asn Met Phe Leu Phe Ser Tyr Pro Glu Ser Gly Tyr Ser Met 615 620 625 ttg tat tct gga cag aaa gga att cag tgt ttc ctg gta gtg gtt gca 2097 Leu Tyr Ser Gly Gln Lys Gly Ile Gln Cys Phe Leu Va l Val Val Ala 630 635 640 cta ctg tgt gta cct tgg atg ctg ctg ttt aaa cca ttg gtc ctt cgc 2145 Leu Leu Cys Val Pro Trp Met Leu Leu Phe Lys Pro Leu Val Leu Arg 645 650 655 655 cgt cag tat ttag agg cat ttg gga act ctc aac ttt ggt ggg 2193 Arg Gln Tyr Leu Arg Arg Lys His Leu Gly Thr Leu Asn Phe Gly Gly 660 665 670 675 atc agg gtg ggc aac gga ccg aca gag gag gat gct gag att att cag 2241 Ile Gly Asn Gly Pro Thr Glu Glu Asp Ala Glu Ile Ile Gln 680 685 690 cat gac cag ctc tcc acc cac tca gag gac gca gac gag ttt gac ttt 2289 His Asp Gln Leu Ser Thr His Ser Glu Asp Ala Asp Glu Phe Asp Phe 695 700 705 ggg gac acc atg gtc cac cag gcc atc cac acc atc gag tac tgc ctg 2337 Gly Asp Thr Met Val His Gln Ala Ile His Thr Ile Glu Tyr Cys Leu 710 715 720 ggc tgc atc tcc aac act gcc tcc tac ttg cgg ctc tgg gcc ctc agc 2385 Gly Cys Ile Ser Asn Thr Ala Ser Tyr Leu Arg Leu Trp Ala Leu Ser 725 730 735 ctc gct cat gcg cac gtg tct gag gtg ctt tgg acc atg gtg atc cac 2433 Leu Ala His Ala His Val Ser Glu Val Leu Trp Thr Met Val Ile His 740 745 750 755 atc ggc ctg agc gtg aag agc ttg gcg gga ggt ttg gtg ctg ttc ttc 2481 Ile Gly Leu Ser Val Lys Ser Leu Ala Gly Gly Leu Val Leu Phe Phe 760 765 act gcc ttt gcc acc ctg acc gtg gcc atc ctc ctg atc atg 2529 Phe Phe Thr Ala Phe Ala Thr Leu Thr Val Ala Ile Leu Leu Ile Met 775 780 785 gag ggc ctc tcg gcc ttt ctc cac gca ctg cgc tta gac tac 2577 Glu Gly Leu Ser Ala Phe Leu His Ala Leu Arg Leu His Trp Val Glu 790 795 800 ttc cag aat aaa ttc tac agc ggg acc ggt ttc aag ttc tta ccc ttc 2625 Phe Gln Asn Lys Phe Tyr Ser Gly Thr Gly Phe Lys Phe Lys Leu Pro Phe 805 810 815 tcc ttc gag cat att cgg gaa ggg aag ttt gaa gag tgagtccctg 2671 Ser Phe Glu His Ile Arg Glu Gly Lys Phe Glu Glu 820 825 830 tgagggccgt gtgcccatgc taccctcccc gcctccctcc acagtgatca gctgttgacc 2731 tctctgcctg ttggttgtga tctgtgggca ccagctcatt cgtgtcaccc tgtctgtgag 2791 tcatttagat agaatagtcc tccttgggtc tcccaccacc cctagctttg tgtgtagtgt 2851 agtgattttc tggctgtcac tcatactcac tgggcaccag ccttgccctc ttagcctcca 2911 tccatccaga cagcccttcc cacctcctgg tggtgagcca gtctgcattc ccacgccatc 2971 ccaaagccct ttcatcttcc ccgtgcattg tagatggaag gagcacccat gccattcacc 3030 <210> 11 <211> 831 <212> PRT <213> Homo sapiens <400> 11 Met Gly Glu Leu Phe Arg Ser Glu Glu Met Thr Leu Ala Gln Leu Phe 1 5 10 15 Leu Gln Ser Glu Ala Ala Tyr Cys Cys Val Ser Glu Leu Gly Glu Leu 20 25 30 Gly Lys Val Gln Phe Arg Asp Leu Asn Pro Asp Val Asn Val Phe Gln 35 40 45 Arg Lys Phe Val Asn Glu Val Arg Arg Cys Glu Glu Met Asp Arg Lys 50 55 60 Leu Arg Phe Val Glu Lys Glu Ile Arg Lys Ala Asn Ile Pro Ile Met 65 70 75 80 Asp Thr Gly Glu Asn Pro Glu Val Pro Phe Pro Arg Asp Met Ile Asp 85 90 95 Leu Glu Ala Asn Phe Glu Lys Ile Glu Asn Glu Leu Lys Glu Ile Asn 100 105 110 Thr Asn Gln Glu Ala Leu Lys Arg Asn Phe Leu Glu Leu Thr Glu Leu 115 120 125 Lys Phe Ile Leu Arg Lys Thr Gln Gln Phe Phe Asp Glu Met Ala Asp 130 135 140 Pro Asp Leu Leu Glu Glu Ser Ser Ser Leu Leu Glu Pro Ser Glu Met 145 150 155 160 Gly Arg Gly Thr Pro Leu Arg Leu Gly Phe Val Ala Gly Val Ile Asn 165 170 175 Arg Glu Arg Ile Pro Thr Phe Glu Arg Met Leu Trp Arg Val Cys Arg 180 185 190 Gly Asn Val Phe Leu Arg Gln Ala Glu Ile Glu Asn Pro Leu Glu Asp 195 200 205 Pro Val Thr G ly Asp Tyr Val His Lys Ser Val Phe Ile Ile Phe Phe 210 215 220 Gln Gly Asp Gln Leu Lys Asn Arg Val Lys Lys Ile Cys Glu Gly Phe 225 230 235 240 Arg Ala Ser Leu Tyr Pro Cys Pro Glu Thr Pro Gln Glu Arg Lys Glu 245 250 255 Met Ala Ser Gly Val Asn Thr Arg Ile Asp Asp Leu Gln Met Val Leu 260 265 270 Asn Gln Thr Glu Asp His Arg Gln Arg Val Leu Gln Ala Ala Ala Lys 275 280 285 285 Asn Ile Arg Val Trp Phe Ile Lys Val Arg Lys Met Lys Ala Ile Tyr 290 295 300 His Thr Leu Asn Leu Cys Asn Ile Asp Val Thr Gln Lys Cys Leu Ile 305 310 315 320 Ala Glu Val Trp Cys Pro Val Thr Asp Leu Asp Ser Ile Gln Phe Ala 325 330 335 Leu Arg Arg Gly Thr Glu His Ser Gly Ser Thr Val Pro Ser Ile Leu 340 345 350 350 Asn Arg Met Gln Thr Asn Gln Thr Pro Pro Thr Tyr Asn Lys Thr Asn 355 360 365 Lys Phe Thr Tyr Gly Phe Gln Asn Ile Val Asp Ala Tyr Gly Ile Gly 370 375 380 Thr Tyr Arg Glu Ile Asn Pro Ala Pro Tyr Thr Ile Ile Thr Phe Pro 385 390 395 400 Phe Leu Phe Ala Val Met Phe Gly Asp Phe Gly His Gly Ile Leu Met 405 410 415 Thr Leu PheAla Val Trp Met Val Leu Arg Glu Ser Arg Ile Leu Ser 420 425 430 Gln Lys Asn Glu Asn Glu Met Phe Ser Thr Val Phe Ser Gly Arg Tyr 435 440 445 Ile Ile Leu Leu Met Gly Val Phe Ser Met Tyr Thr Gly Leu Ile Tyr 450 455 460 Asn Asp Cys Phe Ser Lys Ser Leu Asn Ile Phe Gly Ser Ser Trp Ser 465 470 475 480 Val Arg Pro Met Phe Thr Tyr Asn Trp Thr Glu Glu Thr Leu Arg Gly 485 490 495 Asn Pro Val Leu Gln Leu Asn Pro Ala Leu Pro Gly Val Phe Gly Gly 500 505 510 510 Pro Tyr Pro Phe Gly Ile Asp Pro Ile Trp Asn Ile Ala Thr Asn Lys 515 520 525 Leu Thr Phe Leu Asn Ser Phe Lys Met Lys Met Ser Val Ile Leu Gly 530 535 540 Ile Ile His Met Leu Phe Gly Val Ser Leu Ser Leu Phe Asn His Ile 545 550 555 560 Tyr Phe Lys Lys Pro Leu Asn Ile Tyr Phe Gly Phe Ile Pro Glu Ile 565 570 570 575 Ile Phe Met Thr Ser Leu Phe Gly Tyr Leu Val Ile Leu Ile Phe Tyr 580 585 590 Lys Trp Thr Ala Tyr Asp Ala His Thr Ser Glu Asn Ala Pro Ser Leu 595 600 605 Leu Ile His Phe Ile Asn Met Phe Leu Phe Ser Tyr Pro Glu Ser Gly 610 615 620 Tyr Ser Met LeuTyr Ser Gly Gln Lys Gly Ile Gln Cys Phe Leu Val 625 630 635 640 Val Val Ala Leu Leu Cys Val Pro Trp Met Leu Leu Phe Lys Pro Leu 645 650 655 Val Leu Arg Arg Gln Tyr Leu Arg Arg Lys His Leu Gly Thr Leu Asn 660 665 670 Phe Gly Gly Ily Arg Val Gly Asn Gly Pro Thr Glu Glu Asp Ala Glu 675 680 685 Ile Ile Gln His Asp Gln Leu Ser Thr His Ser Glu Asp Ala Asp Glu 690 695 700 Phe Asp Phe Gly Asp Thr Met Val His Gln Ala Ile His Thr Ile Glu 705 710 715 715 720 Tyr Cys Leu Gly Cys Ile Ser Asn Thr Ala Ser Tyr Leu Arg Leu Trp 725 730 735 Ala Leu Ser Leu Ala His Ala His Val Ser Glu Val Leu Trp Thr Met 740 745 750 Val Ile His Ile Gly Leu Ser Val Lys Ser Leu Ala Gly Gly Leu Val 755 760 765 Leu Phe Phe Phe Phe Thr Ala Phe Ala Thr Leu Thr Val Ala Ile Leu 770 775 780 Leu Ile Met Glu Gly Leu Ser Ala Phe Leu His Ala Leu Arg Leu His 785 790 795 800 Trp Val Glu Phe Gln Asn Lys Phe Tyr Ser Gly Thr Gly Phe Lys Phe 805 810 815 Leu Pro Phe Ser Phe Glu His Ile Arg Glu Gly Lys Phe Glu Glu 820 825 830 <210> 12 <211> 3032 <212> RNA <213> Homo sapiens <400> 12 gggcggcgca ggggcggggc uuuacggacg caagcacguc gaagcgcugc uccuggagcc 60 gcggagggug cggguuuggc ugcggugguu ucuguggcgg uugcuguggc ggaguuugga 120 gguuggagag aaauccaggu acucacuaga cugguaccuu cugccaccau gggggagcuu 180 uuccggagug aagaaaugac acuggcccag cuuuuucuac agucagaggc ugcuuauugu 240 ugugucagug aauuaggaga acuuggaaag guucaguuuc gugacuuaaa uccagaugug 300 aauguuuucc aacggaaauu ugugaaugaa guuagaagau gugaagaaau ggaucgaaag 360 cuucgauuug uugagaaaga gauaagaaaa gcuaacauuc cgauuaugga caccggugaa 420 aacccagagg uucccuuccc ccgggacaug aucgacuuag aggccaauuu ugagaagauu 480 gaaaaugaac ugaaggaaau caacacaaac caggaagcuc ugaagagaaa cuuccuggaa 540 cugaccgaau uaaaauuuau acuucgcaaa acucagcaau uuuuugauga gauggcggau 600 ccagacuugu uggaagaguc cucaucccuc uuggagccaa gugagauggg aagaggcacu 660 ccuuuaagac uuggcuucgu ggcugguguc auuaaccggg agcgcauccc uacuuuugag 720 cgcaugcuuu ggcggguaug ccggggaaau guguuccugc gacaggcuga aaucgagaac 780 ccccuggagg auccugugac uggcgacuac gugcacaagu cuguguuuau cauuuucuuc 840 caaggcgauc a gcugaaaaa cagagucaag aaaaucugug aaggguuccg agccucacuc 900 uaucccuguc cugagacacc acaggagagg aaggaaaugg cuucuggagu gaauaccagg 960 auugaugauc uccaaauggu ucugaaucaa acggaggauc accgccagag gguucugcag 1020 gcagcugcua agaacauccg ugucugguuc aucaaagugc ggaagaugaa ggccaucuau 1080 cacacccuga accugugcaa cauagaugug acucagaaau gcuugauugc agaggucugg 1140 ugcccuguca ccgaccuuga cuccauccag uuugcacuca gaaggggcac ggaacacagu 1200 gguuccacug uaccuuccau uuugaacagg augcagacaa accagacucc cccaaccuau 1260 aacaaaacca acaaguuuac cuauggcuuu cagaacauag uagaugcuua uggaauugga 1320 acuuaccgag agauaaaucc agcuccguau acuauuauca cguucccuuu ucuauuugcu 1380 gugauguuug gagacuucgg ucauggcauu uuaaugaccc uuuuugcugu guggauggua 1440 cugagggaga gccggauccu uucccagaag aaugagaaug agauguuuag cacuguguuc 1500 aguggucgau acauuauuuu auugaugggu guguucucca uguacacugg ccucaucuac 1560 aaugauugcu uuuccaaguc ucuuaauauc uuugggucau ccuggagugu acggccgaug 1620 uuuacuuaua auuggacuga agagacgcuu cgggggaacc cuguucuaca gcugaaccca 1680 gcccucccug gaguguuug g uggaccauac ccuuuuggca uugauccaau uuggaacauu 1740 gcuaccaaua aacugacguu cuugaacucc uuuaagauga agaugucugu uauccuuggu 1800 aucauccaua ugcuguuugg agucagccug agucuguuca accauaucua uuucaagaag 1860 ccccugaaua ucuacuuugg auuuauuccu gaaauaaucu ucaugaccuc uuuguuuggc 1920 uauuugguua uccuuauuuu uuacaagugg acggccuaug augcucauac cucugagaau 1980 gcaccaagcc uucugaucca uuucauaaac auguuccucu uuuccuaccc agagucuggu 2040 uauucaaugu uguauucugg acagaaagga auucaguguu uccugguagu gguugcacua 2100 cuguguguac cuuggaugcu gcuguuuaaa ccauuggucc uucgccguca guauuugagg 2160 agaaagcauu ugggaacucu caacuuuggu gggaucaggg ugggcaacgg accgacagag 2220 gaggaugcug agauuauuca gcaugaccag cucuccaccc acucagagga cgcagacgag 2280 uuugacuuug gggacaccau gguccaccag gccauccaca ccaucgagua cugccugggc 2340 ugcaucucca acacugccuc cuacuugcgg cucugggccc ucagccucgc ucaugcgcac 2400 gugucugagg ugcuuuggac cauggugauc cacaucggcc ugagcgugaa gagcuuggcg 2460 ggagguuugg ugcuguucuu cuucuucacu gccuuugcca cccugaccgu ggccauccuc 2520 cugaucaugg agggccucuc ggcc uuucuc cacgcacugc gcuuacacug gguugaguuc 2580 cagaauaaau ucuacagcgg gaccgguuuc aaguucuuac ccuucuccuu cgagcauauu 2640 cgggaaggga aguuugaaga gugagucccu gugagggccg ugugcccaug cuacccuccc 2700 cgccucccuc cacagugauc agcuguugac cucucugccu guugguugug aucugugggc 2760 accagcucau ucgugucacc cugucuguga gucauuuaga uagaauaguc cuccuugggu 2820 cucccaccac cccuagcuuu guguguagug uagugauuuu cuggcuguca cucauacuca 2880 cugggcacca gccuugcccu cuuagccucc auccauccag acagcccuuc ccaccuccug 2940 guggugagcc agucugcauu cccacgccau cccaaagccc uuucaucuuc cccgugcauu 3000 guagauggaa ggagcaccca ugccauucac cc 3032 <210> 13 <211> 186 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1) .. (186) <400> 13 tat ata acc ggc tca tcc att att ggg ggt ggg gtg aaa gcc ccc aga 48 Tyr Ile Thr Gly Ser Ser Ile Ile Gly Gly Gly Val Lys Ala Pro Arg 1 5 10 15 atc aaa acc aag aac ttg gtt agt att atc ttc tgt gaa gcg gtg gcc 96 Ile Lys Thr Lys Asn Leu Val Ser Ile Ile Phe Cys Glu Ala Val Ala 20 25 30 atc tat ggc atc atc atg gca att gtc atc agc aac atg gct gag cct 144 Ile Tyr Gle Ile Mle Ala Ile Val Ile Ser Asn Met Ala Glu Pro 35 40 45 ttc agt gct acg gag ccc aag gcc att ggc cat cga aac tac 186 Phe Ser Ala Thr Glu Pro Lys Ala Ile Gly His Arg Asn Tyr 50 55 60 <210> 14 <211> 62 <212> PRT <213> Mus musculus <400> 14 Tyr Ile Thr Gly Ser Ser Ile Ile Gly Gly Gly Val Lys Ala Pro Arg 1 5 10 15 Ile Lys Thr Lys Asn Leu Val Ser Ile Ile Phe Cys Glu Ala Val Ala 20 25 30 Ile Tyr Gly Ile Ile Met Ala Ile Val Ile Ser Asn Met Ala Glu Pro 35 40 45 Phe Ser Ala Thr Glu Pro Lys Ala Ile Gly His Arg Asn Tyr 50 55 60 <210> 15 <211> 186 <212> RNA <213> Mus musculus <400> 15 uauauaaccg gcucauccau uauugggggu ggggugaaag cccccagaau caaaaccaag 60 aacuugguua guauuaucuu cugugaagcg guggccaucu auggcaucau cauggcaauu 120 gucaucagca acauggcuga gccuuucagu gcuacggag cccaa <210> 16 <211> 7 <212> PRT <213> Homo sapiens <400> 16 Phe Ser Ala Thr Asp Pro Lys 1 5 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Pro Phe Ser Ala Thr Asp Pro Lys Ala 1 5 <210> 18 <211> 10 <212> PRT <213> Homo sapiens <400> 18 Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala 1 5 10 <210> 19 <211> 20 <212> PRT <213> Homo sapiens <400> 19 Ala Glu Pro Phe Ser Ala Thr Asp Pro Lys Ala Ile Gly His Arg Asn 1 5 10 15 Tyr His Ala Gly 20 <210> 20 <211> 10 <212> PRT <213> Homo sapiens <400> 20 Ile Tyr Gly Ile Ile Met Ala Ile Val Ile 1 5 10 <210> 21 <211> 27 <212> PRT <213> Homo sapiens <400> 21 Phe Ser Ala Thr Asp Pro Lys Ala Ile Gly His Arg Asn Tyr His Ala 1 5 10 15 Gly Tyr Ser Met Phe Gly Ala Gly Leu Thr Val 20 25 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 22 caagaacctg gtcagcatca tc 22 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 23 tgtgaggctg tggccatcta 20 <210> 24 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 24 agactgcggg acg 13 <210> 25 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 25 cgccatgacg gggcta 16 <210> 26 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <221> modified_base <222> 3 <223> n represents a, g, c or t <220> <221> modified_base <222> 6 <223> n represents a, g, c or t <220> <221> modified_base <222> 12 <223> n represents a, g, c or t <400> 26 gcngcntggg gnat 14 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 27 ccccacccac acatatcatc 20 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 28 aatggtgtag cagactcc 18 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 29 catggagtag cctgcatgg 19 <210> 30 <211> 7 <212> PRT <213> Mus musculus <400> 30 Phe Ser Ala Thr Glu Pro Lys 1 5 <210> 31 <211> 9 <212> PRT <213> Mus musculus <400> 31 Pro Phe Ser Ala Thr Glu Pro Lys Ala 1 5 <210> 32 <211> 10 <212> PRT <213> Mus musculus <400> 32 Glu Pro Phe Ser Ala Thr Glu Pro Lys Ala 1 5 10 <210> 33 <211> 20 <212> PRT <213> Mus musculus <400> 33 Ala Glu Pro Phe Ser Ala Thr Glu Pro Lys Ala Ile Gly His Arg Asn 1 5 10 15 Tyr His Ala Gly 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 34 ccccacccac acataacttc 20 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 35 tagacaggcc tacggtga 18 <210> 36 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNA / RNA hybrid molecule <220> <221> modified_base <222> 36 <223> i <220> <221> modified_base <222> 37 <223> i <220> <221> modified_base <222> 41 <223> i <220> <221> modified_base <222> 42 <223> i <220> <221> modified_base <222> 46 <223> i <220> <221> modified_base <222> 47 <223> i <400> 36 cuacuacuac uaggccacgc gtcgactagt acgggnnggg nngggnng 48 <210> 37 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 37 ggagtagcct gcatg 15 <210> 38 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 38 ggccgccatg gtcttca 17 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 39 tgaggagggg tggtctttac 20 <210> 40 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 40 ccaccatggg ggagct 16 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 41 caagtctgga tccgccatc 19 <210> 42 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 42 gccatgacgg ggcta 15 <210> 43 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 43 tagccccgtc atggc 15 <210> 44 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 44 agtgccacag acccc 15 <210> 45 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 45 ggggtctgtg gcact 15 <210> 46 <211> 44 <212> PRT <213> Pinus strobus <400> 46 Val Ala His Gly Tyr Thr Lys Gly Val Gly Leu Ala Ala Glu Ile Ile 1 5 10 15 Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser Ala Thr Asp Pro Lys 20 25 30 Arg Ser Ala Arg Asp Ser His Val Pro Val Leu Ala 35 40 <210> 47 <211> 55 <212> PRT <213> Pinus banksiana <400> 47 Gly Gly Ala Asn Ser Val Ala His Gly Tyr Thr Lys Gly Val Gly Leu 1 5 10 15 Ser Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Ile Phe Ser 20 25 30 Ala Thr Asp Pro Lys Arg Asn Ala Arg Asp Ser His Ile Pro Val Leu 35 40 45 Ala Pro Leu Pro Ile Gly Phe 50 55 <210> 48 <211> 120 <212> PRT <213> Oryza sativa <400> 48 Gly Gly Ala Asn Thr Leu Ala Ala Gly Tyr Ser Lys Gly Thr Gly Leu 1 5 10 15 Ala Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Val Phe Ser 20 25 30 Ala Thr Asp Pro Lys Arg Asn Ala Arg Asp Ser His Val Pro Val Leu 35 40 45 Ala Pro Leu Pro Ile Gly Phe Ala Trp Phe Met Val Gln Leu Ala Thr 50 55 60 Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Thr Thr Gly Pro 65 70 75 80 Ala Val Ile Phe Asn Asn Glu Lys Ala Trp His Asn Gln Trp Ile Phe 85 90 95 Trp Val Gly Pro Phe Val Gly Ala Ala Ile Ala Ala Phe Tyr His Gln 100 105 110 Tyr Ile Leu Arg Ala Gly Pro Ile 115 120 <210> 49 <211> 120 <212> PRT <213> Arabidopsis thaliana <400> 49 Gly Gly Ala Asn Thr Val Ala Asp Gly Tyr Ser Lys Gly Thr Ala Leu 1 5 10 15 Gly Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser 20 25 30 Ala Thr Asp Pro Lys Arg Ser Ala Arg Asp Ser His Ile Pro Val Leu 35 40 45 Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Met Val His Leu Ala Thr 50 55 60 Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser Phe Gly Ala 65 70 75 80 Ala Val Ile Tyr Asn Asn Glu Lys Ala Trp Asp Asp Gln Trp Ile Phe 85 90 95 Trp Val Gly Pro Phe Leu Gly Ala Leu Ala Ala Ala Ala Ala Tyr His Gln 100 105 110 Tyr Ile Leu Arg Ala Ser Ala Ile 115 120 <210> 50 <211> 120 <212> PRT <213> Picea mariana <400> 50 Gly Gly Ala Asn Ser Val Ala His Gly Tyr Thr Lys Gly Val Gly Leu 1 5 10 15 Ser Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser 20 25 30 Ala Thr Asp Pro Lys Arg Asn Ala Arg Asp Ser His Val Pro Val Leu 35 40 45 Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Met Val His Leu Ala Thr 50 55 60 Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser Phe Gly Ala 65 70 75 80 Ala Val Ile Tyr Gly His Gln Lys Ile Trp Asp Glu His Trp Ile Phe 85 90 95 Trp Val Gly Pro Phe Leu Gly Ala Ala Gly Ala Ala Ala Ala Tyr His Gln 100 105 110 Tyr Ile Leu Arg Ala Gly Ala Ile 115 120 <210> 51 <211> 120 <212> PRT <213> Samanea saman <400> 51 Gly Gly Ala Asn Thr Leu Ser Asp Gly Tyr Ser Thr Gly Thr Gly Leu 1 5 10 15 Gly Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser 20 25 30 Ala Thr Asp Pro Lys Arg Ser Ala Arg Asp Ser His Val Pro Val Leu 35 40 45 Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Met Val His Leu Ala Thr 50 55 60 Ile Pro Val Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser Leu Gly Ala 65 70 75 80 Ala Val Ile Phe Asn Gln Gln Lys Ala Trp Asp Asp His Trp Ile Phe 85 90 95 Trp Val Gly Pro Phe Ile Gly Ala Ala Ile Ala Ala Phe Tyr His Gln 100 105 110 Phe Ile Leu Arg Ala Gly Ala Ala 115 120 <210> 52 <211> 120 <212> PRT <213> Mesembryanthemum crystallinum <400> 52 Gly Gly Ala Asn Glu Leu Ser Val Gly Tyr Ser Lys Gly Thr Gly Leu 1 5 10 15 Ala Ala Glu Ile Ile Gly Thr Phe Val Leu Val Tyr Thr Val Phe Ser 20 25 30 Ala Thr Asp Pro Lys Arg Ser Ala Arg Asp Ser His Val Pro Val Leu 35 40 45 Ala Pro Leu Pro Ile Gly Phe Ala Val Phe Met Val His Leu Ala Thr 50 55 60 Ile Pro Ile Thr Gly Thr Gly Ile Asn Pro Ala Arg Ser Leu Gly Ala 65 70 75 80 Ala Val Ile Tyr Asn Asn Asn Lys Ala Trp Asp Asp Gln Trp Ile Phe 85 90 95 Trp Val Gly Pro Phe Ile Gly Ala Ala Ile Ala Ala Ile Tyr His Gln 100 105 110 Tyr Ile Leu Arg Ala Gly Phe Ile 115 120

[0230]

[Sequence List Free Text]

SEQ ID NO: 22: Synthetic DNA SEQ ID NO: 23: Synthetic DNA SEQ ID NO: 24: Synthetic DNA SEQ ID NO: 25: Synthetic DNA SEQ ID NO: 26: Synthetic DNA SEQ ID NO: 26: n represents a, g, c or t (location: 3 ). SEQ ID NO: 26: n represents a, g, c, or t (location: 6). SEQ ID NO: 26: n represents a, g, c, or t (location: 1
2).

SEQ ID NO: 27: Synthetic DNA SEQ ID NO: 28: Synthetic DNA SEQ ID NO: 29: Synthetic DNA SEQ ID NO: 34: Synthetic DNA SEQ ID NO: 35: Synthetic DNA SEQ ID NO: 36: Synthetic DNA SEQ ID NO: 36: DNA / RNA binding molecule

SEQ ID NO: 37: Synthetic DNA SEQ ID NO: 38: Synthetic DNA SEQ ID NO: 39: Synthetic DNA SEQ ID NO: 41: Synthetic DNA SEQ ID NO: 42: Synthetic DNA SEQ ID NO: 43: Synthetic DNA SEQ ID NO: 44: Synthetic DNA SEQ ID NO: 45: Synthetic DNA

[Brief description of the drawings]

FIG. 1 shows the basic structure of V-ATPase.
That is, in a tumor cell, subunit c which is a 16 kDa proteolipid, subunit c which is a 21 kDa proteolipid, and subunit a which is a 116 kDa proteolipid are extracellular membranes in a tumor cell. Is exposed.

Fig. 2. Normal thyroid cell culture using KTC-3 antibody (NTC-
It is a photograph which shows the fluorescent antibody staining of 1).

FIG. 3. Human papillary thyroid carcinoma culture (TPC) using KTC-3 antibody
1 is a photograph showing the fluorescent antibody staining of 1).

FIG. 4 is a photograph showing immunohistochemical staining of human papillary thyroid carcinoma using a KTC-3 antibody.

FIG. 5 is a photograph showing immunohistochemical staining of undifferentiated human thyroid cancer using a KTC-3 antibody.

FIG. 6 is a photograph showing localization of a tumor antigen in a human papillary thyroid cancer cell line (TPC-1).

FIG. 7: KTC against human papillary thyroid carcinoma culture (TPC-1)
3 is a photograph showing the cell killing effect of Anti-3 antibody (trypan blue staining).

FIG. 8 shows the results of separation of KTC-3 antibody using a Protein A Sepharose column.

FIG. 9: KTC against human papillary thyroid cancer cell line (TPC-1)
4 is a photograph showing the cell-killing effect of the Fab portion of Anti-3 antibody (after 24 hours of culture).

FIG. 10: K against human papillary thyroid cancer cell line (TPC-1)
4 is a photograph showing the cell killing effect (after 72 hours of culture) of the Fab portion of the TC-3 antibody.

FIG. 11 is a photograph showing the reactivity of a KTC-3 antibody with a peptide of the present invention.

FIG. 12 is a photograph showing the reactivity of a KTC-3 antibody with a peptide of the present invention.

FIG. 13 is a view showing the results of analysis of the amino acid sequence of the SSY tumor antigen by a profile method (hydrophobicity plotting).

FIG. 14 is a photograph showing the expression results of SSY antigen in cancer tissues and normal tissues.

FIG. 15 is a photograph showing the expression results of SSY antigen in cancer tissues and normal tissues.

FIG. 16 is a photograph showing the expression result of SSY antigen in a cancer tissue.

FIG. 17 is a photograph showing a result of detecting a cancer by scintigram.

FIG. 18 is a photograph showing the expression result of SSY antigen in various tumor materials.

FIG. 19 is a photograph showing the expression results of the SSY antigen and the c-subunit and a-subunit of V-ATPase in breast cancer.

FIG. 20 shows the antitumor effect of an anti-c subunit antibody on mouse erythroleukemia cells (EL-4).

FIG. 21. Bafilomycin A1 which is a V-ATPase inhibitor
FIG. 4 is a graph showing the inhibition of CHO cell growth in Example 1.

FIG. 22. Bafilomycin B1, a V-ATPase inhibitor
FIG. 4 is a graph showing the inhibition of CHO cell growth in Example 1.

FIG. 23: CHO of hygrolysin which is a V-ATPase inhibitor
It is a figure which shows cell growth suppression.

FIG. 24. Concanamicin B which is a V-ATPase inhibitor
It is a figure which shows CHO cell growth suppression.

FIG. 25 is a graph showing suppression of CHO cell growth by O-methylconkanamycin B, a V-ATPase inhibitor.

FIG. 26. CHO of leucanisidine, a V-ATPase inhibitor
It is a figure which shows cell growth suppression.

FIG. 27: CH of destruxin E, a V-ATPase inhibitor
It is a figure which shows O cell proliferation suppression.

FIG. 28 is a diagram showing the inhibition of PC12 cell growth by Destraxin B and Destraxin E, which are V-ATPase inhibitors.

FIG. 29 is a diagram showing the inhibition of PC12 cell growth by metacycloprodigiosin, prodigiosin and prodigiosin 25-C, which are H + / Cl- symporters (V-ATPase uncouplers).

FIG. 30A: V-ATPase inhibitors (bafilomycin A1, bafilomycin B1, hygrolysin and leucanidine)
FIG. 2 is a view showing a chemical structure of

FIG. 30B: V-ATPase inhibitors (conkanamycin B and o
FIG. 3 shows the chemical structure of -Methylconcanamycin B).

FIG. 30C shows the chemical structures of V-ATPase inhibitors (Destraxin B and Destraxin E).

FIG. 30D: H + / K + -ATPase inhibitor (prodigiosins)
FIG. 2 is a view showing a chemical structure of

FIG. 31 is a graph showing the inhibition of proliferation of TPC-1 by rabeprazole sodium, which is an H + / K + -ATPase inhibitor.
(72 hours after administration of rabeprazole sodium 2 μg / ml)

FIG. 32 is a graph showing the inhibition of growth of rabeprazole sodium on TPC-1. (Rabeprazole sodium
72 hours after administration of 20 μg / ml)

FIG. 33. TPC- without adding rabeprazole sodium
It is a figure which shows 1 cell (control). (After 72 hours of culture)

FIG. 34 is a graph showing the inhibition of rabeprazole sodium on the growth of EL-4 cells (rabeprazole sodium
72 hours after administration of 2 μg / ml).

FIG. 35 is a graph showing the inhibition of rabeprazole sodium on the growth of EL-4 cells (rabeprazole sodium
72 hours after administration of 20 μg / ml).

FIG. 36. EL-4 without adding rabeprazole sodium
It is a figure which shows a cell (control). (After 72 hours of culture)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61P 35/00 4C084 48/00 C07K 7/06 4C085 A61P 35/00 7/08 4C087 C07K 7 / 06 14/705 4H045 7/08 16/28 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 9/99 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z 9/99 33/50 Z C12Q 1/68 33/574 A G01N 33/15 33/577 B 33/50 C12P 21/08 33/574 C12N 15/00 ZNAA 33/577 A61K 37/64 / (72) Inventor Nobutaka Suzuki 10-22 Oyamacho, Kanazawa City, Ishikawa Prefecture (72) Inventor Masaaki Yamaguchi 10-6 Showacho, Kanazawa City, Ishikawa Prefecture (72) Inventor Nobuo Yamaguchi 61, Okidai, Uchinada, Hebei-gun, Ishikawa Prefecture (72) Katsuharu Okuma 1-22-14, Hoshima-cho, Kanazawa, F-term F-term (reference) QR08 QR32. AA17 BB22 DD63 DD88 4C087 AA01 AA02 BB37 ZB26 ZB27 4H045 AA10 AA11 AA30 BA10 BA14 BA15 BA17 BA21 CA41 DA75 DA76 DA86 EA28 EA51 FA34 FA72 FA74

Claims (49)

[Claims] 1. A diagnostic agent for a tumor, comprising as an active ingredient at least one selected from the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least one amino acid sequence in which at least one amino acid sequence is deleted, substituted or added in a sequence containing at least the first amino acid sequence or a partial sequence thereof and which reacts specifically with tumor (c) represented by SEQ ID NO: 8 (D) an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, wherein one or several amino acids are deleted, substituted or added, and (E) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) One or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof , A protein comprising a substituted or added amino acid sequence and reacting tumor-specifically 2. The method according to claim 1, wherein the tumor is thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The diagnostic agent according to claim 1. 3. A prophylactic or therapeutic agent for a tumor, comprising as an active ingredient at least one selected from the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least one amino acid sequence in which at least one amino acid sequence is deleted, substituted or added in a sequence containing at least the first amino acid sequence or a partial sequence thereof and which reacts specifically with tumor (c) represented by SEQ ID NO: 8 (D) an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, wherein one or several amino acids are deleted, substituted or added, and (E) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) One or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof , A protein comprising a substituted or added amino acid sequence and reacting tumor-specifically 4. The method according to claim 1, wherein the tumor is thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The prophylactic or therapeutic agent according to claim 3, wherein 5. A diagnostic agent for a tumor, comprising as an active ingredient an antibody against any one of the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least one amino acid sequence in which at least one amino acid sequence is deleted, substituted or added in a sequence containing at least the first amino acid sequence or a partial sequence thereof and which reacts specifically with tumor (c) represented by SEQ ID NO: 8 (D) an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, wherein one or several amino acids are deleted, substituted or added, and (E) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) One or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof , A protein comprising a substituted or added amino acid sequence and reacting tumor-specifically 6. The tumor may be thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The diagnostic agent according to claim 5, wherein 7. A preventive or therapeutic agent for a tumor, comprising as an active ingredient an antibody against any one of the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least one amino acid sequence in which at least one amino acid sequence is deleted, substituted or added in a sequence containing at least the first amino acid sequence or a partial sequence thereof and which reacts specifically with tumor (c) represented by SEQ ID NO: 8 (D) an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, wherein one or several amino acids are deleted, substituted or added, and (E) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) One or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof , A protein comprising a substituted or added amino acid sequence and reacting tumor-specifically 8. The tumor may be thyroid cancer, breast cancer, stomach cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The prophylactic or therapeutic agent according to claim 7. 9. At least one selected from the group consisting of the following DNAs (a) to (f) or an antisense DNA thereof:
Diagnostic agents for tumors. (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof (b) Hybridizing with the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof under stringent conditions, and being tumor-specific (C) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 7 or a partial sequence thereof (d) DNA comprising the nucleotide sequence represented by SEQ ID NO: 7 or a partial sequence thereof under stringent conditions DNA encoding a protein that hybridizes and reacts specifically with tumor (e) DNA consisting of the base sequence represented by SEQ ID NO: 10 or a partial sequence thereof (f) Base sequence represented by SEQ ID NO: 10 or a part thereof DNA encoding a protein that hybridizes to the sequence under stringent conditions and reacts specifically with tumor 10. At least one selected from the group consisting of RNAs of the following (a) to (f) or an antisense RN thereof:
A diagnostic agent for a tumor, including A. (a) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 or a partial sequence thereof (b) Hybridizing with the nucleotide sequence represented by SEQ ID NO: 3 or a partial sequence thereof under stringent conditions, and being tumor-specific (C) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 9 or a partial sequence thereof (d) RNA comprising a nucleotide sequence represented by SEQ ID NO: 9 or a partial sequence thereof under stringent conditions RNA encoding a protein that hybridizes and reacts specifically with tumor (e) RNA consisting of the base sequence represented by SEQ ID NO: 12 or a partial sequence thereof (f) Base sequence represented by SEQ ID NO: 12 or a part thereof RNA encoding a protein that hybridizes with the sequence under stringent conditions and reacts specifically with tumor 11. The DNA according to claim 9 or claim 1.
A diagnostic agent for a tumor comprising a recombinant vector containing the RNA of 0 or a transformant containing the recombinant vector. 12. The tumor may be thyroid, breast, gastric, esophageal, oral, colon, rectal, anal, pancreatic, lung,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The diagnostic agent according to any one of claims 9 to 11. 13. An antisense DN or at least one selected from the group consisting of the following DNAs (a) to (f):
A therapeutic agent for tumor, including A. (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof (b) Hybridizing with the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof under stringent conditions, and being tumor-specific (C) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 7 or a partial sequence thereof (d) DNA comprising the nucleotide sequence represented by SEQ ID NO: 7 or a partial sequence thereof under stringent conditions DNA encoding a protein that hybridizes and reacts specifically with tumor (e) DNA consisting of the base sequence represented by SEQ ID NO: 10 or a partial sequence thereof (f) Base sequence represented by SEQ ID NO: 10 or a part thereof DNA encoding a protein that hybridizes to the sequence under stringent conditions and reacts specifically with tumor 14. An antisense RN or at least one selected from the group consisting of the following RNAs (a) to (f):
A therapeutic agent for tumor, including A. (a) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 or a partial sequence thereof (b) Hybridizing with the nucleotide sequence represented by SEQ ID NO: 3 or a partial sequence thereof under stringent conditions, and being tumor-specific (C) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 9 or a partial sequence thereof (d) RNA comprising a nucleotide sequence represented by SEQ ID NO: 9 or a partial sequence thereof under stringent conditions RNA encoding a protein that hybridizes and reacts specifically with tumor (e) RNA consisting of the base sequence represented by SEQ ID NO: 12 or a partial sequence thereof (f) Base sequence represented by SEQ ID NO: 12 or a part thereof RNA encoding a protein that hybridizes with the sequence under stringent conditions and reacts specifically with tumor 15. The DNA according to claim 13 or claim 1.
A therapeutic agent for a tumor, comprising the recombinant vector containing the RNA according to 4, or a transformant containing the recombinant vector. 16. The tumor may be thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The therapeutic agent according to any one of claims 1 to 15. 17. A tumor-specific antigen is detected by reacting the DNA according to claim 9, the RNA according to claim 10, or the recombinant vector or transformant according to claim 11 with a body fluid or a tumor cell. how to. 18. A leukocyte which reacts with at least one selected from the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least one amino acid sequence in which at least one amino acid sequence is deleted, substituted or added in a sequence containing at least the first amino acid sequence or a partial sequence thereof and which reacts specifically with tumor (c) represented by SEQ ID NO: 8 (D) an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, wherein one or several amino acids are deleted, substituted or added, and (E) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) One or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof , A protein comprising a substituted or added amino acid sequence and reacting tumor-specifically 19. A leukocyte which binds to an antibody against any one of the following proteins (a) to (f): (a) No. 1 to 85 of the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least the amino acid sequence of the th or a partial sequence thereof (b) the amino acid sequence represented by SEQ ID NO: 2
A protein comprising at least one amino acid sequence in which at least one amino acid sequence is deleted, substituted or added in a sequence containing at least the first amino acid sequence or a partial sequence thereof and which reacts specifically with tumor (c) represented by SEQ ID NO: 8 (D) an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, wherein one or several amino acids are deleted, substituted or added, and (E) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof (f) One or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof , A protein comprising a substituted or added amino acid sequence and reacting tumor-specifically 20. A pharmaceutical composition comprising the leukocyte according to claim 18 as an active ingredient. 21. An agent for preventing or treating a tumor, comprising the leukocyte according to claim 18 as an active ingredient. 22. The tumor, wherein the tumor is thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma 22. The prophylactic or therapeutic agent according to claim 21. 23. A tumor-specific antigen peptide of the following (a) or (b): (a) a tumor-specific antigen peptide consisting of an amino acid sequence represented by Phe Ser Ala Thr Glu Pro Lys; (b) a tumor containing at least the amino acid sequence represented by Phe Ser Ala Thr Glu Pro Lys, and being tumor-specifically expressed Specific antigen peptide. 24. A tumor-specific antigen peptide which comprises at least the 49th to 55th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 14 and is specifically expressed in a tumor. 25. A tumor-specific antigen peptide of the following (a) or (b): (a) a tumor-specific antigen peptide consisting of any of the amino acid sequences represented by SEQ ID NOS: 31 to 33; (b) one or several amino acids deleted in any of the amino acid sequences represented by SEQ ID NOS: 31 to 33 , A tumor-specific antigen peptide comprising a substituted or added amino acid sequence and expressed in a tumor-specific manner. 26. A nucleic acid encoding a protein comprising the peptide according to any one of claims 23 to 25. 27. The nucleic acid is a DNA of the following (a) or (b):
27. The nucleic acid according to claim 26, which is the RNA of (c) or (d). (A) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 13 (b) hybridizing with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 13 under stringent conditions, and a peptide that is specifically expressed in tumor Encodes a protein containing
DNA (c) RNA consisting of the nucleotide sequence represented by SEQ ID NO: 15 (d) Peptide that hybridizes with RNA consisting of the nucleotide sequence represented by SEQ ID NO: 15 under stringent conditions, and is specifically expressed in a tumor Encodes a protein containing
RNA 28. A recombinant vector containing the nucleic acid according to claim 26 or 27. A transformant comprising the recombinant vector according to claim 28. 30. An antibody against the peptide according to any one of claims 23 to 25. 31. The antibody according to claim 30, wherein the antibody is a monoclonal antibody or a polyclonal antibody or a fragment thereof. 32. A leukocyte which reacts with the peptide according to any one of claims 23 to 25 or the antibody according to claim 30 or 31. 33. A pharmaceutical composition comprising the peptide according to any one of claims 23 to 25, the antibody according to claim 30 or 31, or the leukocyte according to claim 33 as an active ingredient. 34. An agent for preventing or treating a tumor, comprising as an active ingredient a proton pump inhibitor or a proton pump uncoupler (excluding bafilomycin, lansoprazole and BE-18591). 35. An agent for preventing or treating a tumor, comprising a V-ATPase inhibitor (excluding bafilomycin) as an active ingredient. 36. The V-ATPase inhibitor, wherein EDTA, EGTA, conkanamycin A, conkanamycin B, O-methylconkanamycin B, N-ethylmaleimide, N, N'-dicyclohexylcarbodiimide, 7-chloro-4- Nitrobenz-2-oxa-1,3-diazole, suramin, verapamil, (2Z, 4E) -5
-(5,6-Dichloro-2-indolyl) -2-methoxy-N- (1,2,2,
6,6-pentamethylpiperidin-4-yl) -2,4-pentadienamide, OST-766, 5- (5,6-dichloro-2-indolyl) -2
-Methoxy-2,4-pentadienamide, NO ₃ , 3′-O- (4-benzoyl) benzoyladenosine 5′-triphosphate, destraxin B, destraxin E, leucanisidine, hygrolizine, bisphosphonate tiludronate, diethyl Pyrocarbonate, dibromoacetophenone, p-chloromercurybenzoic acid, quercetin, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanostilbene-2,2 ' 36. The prophylactic or therapeutic agent according to claim 35, which is at least one selected from the group consisting of -disulfonic acid, N-bromosuccinimide and benzoyltaxinine K. 37. A prophylactic or therapeutic agent for a tumor, comprising an H + / K + -ATPase inhibitor (excluding lansoprazole) as an active ingredient. 38. The preventive or therapeutic agent for a tumor according to claim 39, wherein the H + / K + -ATPase inhibitor is a benzimidazole derivative. 39. A benzimidazole derivative comprising rabeprazole sodium, omeprazole, pantoprazole, reminoprazole, 2-dimethylamino-4,5-dihydrothiazolo [4,5: 3,4] pyridol [1,2-a ] Benzimidazole and 2-aryl-4,5-dihydro-1H-thieno [3,2-e]
39. The prophylactic or therapeutic agent according to claim 38, which is at least one selected from the group consisting of benzimidazole. 40. The method according to claim 14, wherein the H + / K + -ATPase inhibitor is (±) -5-methoxy-2-[[(4-methoxy-3,5-dimethylpyrid-2-yl) methyl] sulfinyl] -1H-imidazo ( 4,5-b) pyridine, 3-
Amino-5-methyl-2- (2-methyl-3-thienyl) imidazo [1,
2-a] thieno [3,2-c] pyridine, 1-aryl-3-substituted pyrrolo
[3,2-c] quinoline, 3-butyryl-8-methoxy-4-[(2-thiophenyl) amino] quinoline, 2-[(3,5-dimethyl-4-methoxypyridyl) alkyl] -benzothiazolidine, 3-butyryl-4- [R-1-methylbenzylamino] -8-ethoxy-1,7-
38. The prophylactic or therapeutic agent according to claim 37, which is at least one selected from the group consisting of naphthyridine and pentagalloylglucose. 41. An agent for preventing or treating a tumor, comprising as an active ingredient a V-ATPase uncoupler containing H + / Cl- symporter (excluding BE-18591). 42. The prophylactic or therapeutic agent according to claim 41, wherein the V-ATPase uncoupling agent containing an H + / Cl- symporter is a prodigiosin (excluding BE-18591). 43. The prodigiosins are prodigiosin, metacycloprodigiosin, prodigiosin 25-C,
43. The prophylactic or therapeutic agent according to claim 42, which is at least one selected from the group consisting of cycloprodigiosin and cycloprodigiosin hydrochloride. 44. The method according to claim 41, wherein the V-ATPase uncoupling agent containing the H + / Cl- symporter is at least one selected from the group consisting of bepridil, manganese tetraphenylporphine, and thallium chloride. Therapeutic agent. 45. The tumor, wherein the tumor is thyroid cancer, breast cancer, gastric cancer, esophageal cancer, oral cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, lung cancer,
Kidney cancer, bladder cancer, ovarian cancer, uterine cancer, vulvar cancer, skin cancer, melanoma, central nerve tumor, peripheral nerve tumor, gingival cancer, pharyngeal cancer, jaw cancer, mediastinal tumor, liver cancer, bile duct cancer, gallbladder cancer, renal pelvis At least one selected from the group consisting of tumor, ureter cancer, testicular tumor, prostate cancer, choriocarcinoma, fallopian tube cancer, vaginal cancer, sarcoma, leukemia, erythroleukemia, multiple myeloma, malignant lymphoma and carcinosarcoma The prophylactic or therapeutic agent according to any one of claims 34 to 44. 46. A functional antitumor composition comprising the peptide or protein according to any one of claims 1 and 23 to 25. 47. A functional antitumor composition comprising a plant containing the amino acid sequence represented by Phe Ser Ala Thr Asp Pro Lys. 48. A functional antitumor composition comprising aquaporin containing an amino acid sequence represented by Phe Ser Ala Thr Asp Pro Lys or a fragment thereof. 49. The fragment of aquaporin according to SEQ ID NOs: 46-5
49. The composition according to claim 48, which is at least one selected from those represented by 2.