JP2002112780A - Human bladder cancer antigen - Google Patents

Abstract

(57) [Summary] [Problem] Diagnosis of various cancers and tumors including bladder cancer
To provide a human bladder cancer antigen which can be applied to therapy, a gene encoding the same, and the like. SOLUTION: A total of 4.0 bladder cancer cell cDNA libraries prepared by preparing cDNA using mRNA obtained from a bladder cancer cell line, introducing the cDNA into a λ phage vector and infecting Escherichia coli. × 10
^Five λ phage cDNA clones were screened one by one using bladder cancer patient serum, and reacted only with bladder cancer patient serum, did not react with healthy human serum, and were highly expressed in bladder cancer cells and tissues. The antigen KU-BL-50 is produced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel human bladder cancer antigen useful for diagnosis and immunotherapy of various cancers including bladder cancer, and uses thereof.

[0002]

2. Description of the Related Art Immunotherapy has been expected for a long time, and various attempts have been made, but it has not yet achieved a sufficient antitumor effect. Conventionally, immunotherapy of cancer has been mainly performed by non-specific immunotherapy. However, in recent years, it has been revealed in human melanoma that T cells play an important role in tumor rejection in vivo, and furthermore, MAGE- 1 antigen (Scie
254, 1643-7, 1991) was reported by the Belgian group in 1991, and cytotoxic T cells (CT
L: efforts are being made to isolate T cell-recognizing tumor antigens that can induce cytotoxic T lymphocytes and to determine MHC class I-restricted epitopes. The present inventors isolated a CD8 + T cell recognition antigen by a cDNA expression cloning method using melanoma tumor-reactive T cells (Proc. Natl.
Acad. Sci. USA 91, 3515-3519, 1994, Proc. Natl. Ac
ad. Sci. USA 91, 6458-6462, 1994, J. Exp. Med. 18
0, 347-352, 1994; J. Immunol. 154, 3961-3968, 199
5, Immunologic Res. 16, 313-340, 1997), reported that specific immunotherapy using this antigen had antitumor effects on some melanomas (Micr).
obiology Immunology 42, 803-813, 1998, Kawakami,
Y., PF Robbins, RF. Wang. Et al. Identification
of Melanoma antigens by T lymphocytes and theiruse
in the immunotherapy of cancer.In Principle and
Practice of Oncology. Update. V. DeVita, S. Hellma
n, SARosenberg eds.JBLippincott Co. Philadelph
ia, p1-20, 1996, Nature Med. 4, 321, 1998).

[0003] As a treatment method for bladder cancer, a treatment method combining endoscopic resection (TUR-BT) and intravesical infusion therapy for superficial bladder cancer and a treatment method for locally invasive bladder cancer have been conventionally used. A curative treatment method using a combination of radical cystectomy and further chemotherapy or radiation treatment is known. However, for the treatment of metastatic bladder cancer, me
Combination chemotherapy (M-VAC) and radiation therapy using thotrexate, vinbrastine, adriamycin, and cisplatin, as well as combination therapy have been performed, but no long-term treatment is expected and long-term treatment is still not possible Pessimistic.

[0004] In the treatment of bladder cancer, intravesical instillation of BCG is widely used for treatment of bladder intraepithelial neoplasia and prevention of recurrence of superficial bladder cancer, and its effectiveness has been confirmed. The mechanism of this antitumor effect has not been completely elucidated, but has been suggested to be due to an immune response. After intravesical instillation of BCG, a marked increase in mononuclear cells was observed in the bladder tumor tissue (lymphocyte dominance), and an increase in the proportion of helper T cells was observed in the tumor tissue compared to before BCG use. (J. Uro
l. 144, 53-8, 1990). In addition, interleukin (IL) -1, IL-2, IL-6, T
Detection of cytokines such as NF-α and interferon γ (IFN-γ) suggests that the immune response is related to the mechanism of the antitumor effect of BCG. These facts suggest that the bladder cancer antigen may be effective for immunotherapy as a treatment for metastatic bladder cancer. However, at present, an antigen highly specific for bladder cancer has not been isolated, and immunotherapy using an antigen vaccine only for bladder cancer expressing cancer antigens isolated from other cancer types Was just happening.

Conventionally, CTLs have been used as isolations of many tumor antigens.
However, isolation of tumor antigens from carcinomas other than melanoma is difficult due to the necessity of tumor cell lines and establishment of CTL. In addition, in order to enhance the effects of immunotherapy, it is thought that a therapeutic method in which many peptides are mixed is effective, but in order to establish it, it is necessary to isolate a large number of antigens. The cDNA expression cloning method is as follows.
There was a problem that a lot of labor and time were required for isolation of one antigen.

[0006] In 1995, a group of Pfreundschuh of Germany and Old et al. Of the United States recognized a cancer recognized by an antibody in the serum of a cancer patient as applicable to carcinomas that do not necessarily require tumors and CTLs and have difficulty establishing cell lines. SEREX method (serological identificatio
n of reconbinant cDNA expression cloning; Proc. Nat
l. Acad. Sci. USA 92, 11810-11813, 1995). Although many tumor antigens have been isolated by this method, some of the antigens isolated using this method include CTL
Since MAGE-1 and tyrosinase-inducing antigens are found, they are pointed out to be useful as a method for detecting an antigen recognized by cell-mediated immunity. Also,
It has been reported that a cancer antigen recognized by a patient IgG antibody in melanoma, renal cancer, esophageal cancer, colorectal cancer, lung cancer and the like was isolated using the above method (Int. J. Cancer 72, 9).
65-971, 1997, Cancer Res. 58, 1034-1041, 1998, In
tJ Cancer 29, 652-658, 1998, Int.J. Oncol. 14,
703-708, 1999, Cancer Res. 56, 4766-4772, 1996, Hu
m. Mol. Genet 6, 33-39, 1997). These antigens include C
T (cancer-testis) antigen (SSX2 / HOM-MEL)
-40, NY-ESO-1, SCP-1, CT7, etc.), differentiation antigens (galectin-4 / NY-CO-
27) and important antigens such as mutant antigens (such as p53). Among these antigens, especially NY-ESO-1 is HLA
-A2 restricted epitopes have been determined. Also, S
Among the antigens isolated using the EREX method, elF-
Some molecules are highly expressed in cancer, such as 4γ and HER2 / neu, and these molecules may be related to canceration.
Molecules highly expressed in such cancers may not only be applied to therapy, but may also be diagnostic markers and markers that can predict recurrence and prognosis.

At present, few tumor antigens have been identified in bladder cancer. Recently, KIA is also expressed in normal tissues as an antigen recognized directly by CTL in bladder tumors
Mutation of the A0205 antigen identified an antigenic molecule [MHC class I (HLA-B4403) restricted] for the first time (J. Immunol. 160, 6188-94,
1998). However, this molecule is found in 1 in 60 bladder cancer tissues.
No expression was observed in any of the examples, and it is considered that only one individual had changed. Further, by screening a cDNA library prepared from the T24 bladder tumor cell line using the serum of a hepatocellular carcinoma patient, the gene isolated by the SEREX method using the previous melanoma cell line, KOC3 highly expressed in
The gene was isolated. However, these molecules cannot be said to be tumor antigens with high specificity for bladder cancer, and few antigens are still applicable to immunotherapy of bladder cancer. Regarding the diagnosis of bladder cancer, no highly specific antigen such as PSA of prostate cancer has been found yet.

[0008]

At present, cancer is the leading cause of death.
Despite advances in treatment, many advanced cancers cannot yet be treated. To improve this, new early diagnostics and treatments need to be developed. An object of the present invention is to provide a human bladder cancer antigen which can be applied to diagnosis and treatment of various cancers including bladder cancer and tumors, a gene encoding the same, and the like.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, prepared cDNA using mRNA obtained from a bladder cancer cell line, and converted this cDNA into a λ phage vector. A bladder cancer cell cDNA library prepared by transfection and infection of E. coli, a total of 4.0 × 10 ⁵ λ phage cDNA clones,
A bladder cancer antigen KU-BL which is screened one by one using bladder cancer patient serum, reacts only with bladder cancer patient serum, does not react with healthy human serum, and is highly expressed in bladder cancer cells and tissues
-50 was found, and the present invention was completed.

That is, the present invention relates to a DNA encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence shown in SEQ ID NO: 2; A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having immunity-inducing activity (Claim 1)
It hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, a DNA containing a part or all of these sequences (claim 2), or the DNA described in claim 2 under stringent conditions. And DNA encoding a protein having an immunity-inducing activity (Claim 3), a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (Claim 4), or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2. Proteins comprising an amino acid sequence wherein amino acids have been deleted, substituted or added, and having an immunity-inducing activity (claim 5) or a part of the protein according to claim 4 or 5; (Claim 6).

[0011] The present invention also provides a fusion protein or fusion peptide in which a protein or a peptide according to claim 4 or 5 is linked to a marker protein and / or a peptide tag (claim 7). An antibody that specifically binds to the protein of claim 4 or 5 or the peptide of claim 6 (claim 8), or the antibody is a monoclonal antibody (claim 9). Or a recombinant protein or peptide to which the antibody according to claim 8 or 9 specifically binds (claim 1).
0).

Further, the present invention provides any one of claims 4 to 6,
Alternatively, a host cell comprising an expression system capable of expressing the protein or peptide according to claim 10 (claim 11), or any one of claims 4 to 6, or encoding the protein or peptide according to claim 10 A non-human animal (claim 12) characterized in that the gene function is deficient on the chromosome, or overexpressing the protein or peptide according to any one of claims 4 to 6 or claim 10. The present invention relates to a non-human animal according to claim 12 or claim 13 wherein the non-human animal is a mouse or a rat.

[0013] The present invention also provides a test substance;
A method of screening for a substance that promotes or suppresses immunity-inducing activity, comprising using the protein or peptide according to claim 10 or T-cells and measuring and evaluating immunity-inducing activity in said T-cells (claim Item 15)
And a test substance, the host cell according to claim 11, and a T cell, wherein the immunity induction activity in the T cell is measured and evaluated. (16) The host cell according to (11), wherein the vector expressing HLA and the vector expressing the test polypeptide are both transfected;
A method for screening for a substance that promotes or suppresses immunity-inducing activity using T cells and measuring and evaluating the immunity-inducing activity in the T cells (claim 17). H
A method for screening for a substance that promotes or suppresses immunity-inducing activity, comprising measuring and evaluating the immunity-inducing activity of T cells using the host cell according to claim 11 having LA expression ability and T cells (claim) 18) and claims 12 to
Administering a test substance to the non-human animal according to any of 14 above,
A method for screening for a substance that promotes or suppresses immunity-inducing activity, characterized by measuring and evaluating immunity-inducing activity in T cells of the non-human animal (claim 19), and measuring and evaluating immunity-inducing activity by interferon in T cells The measurement / evaluation of γ activity, wherein the γ activity is measured and evaluated.
9. A method for screening a substance for promoting or suppressing immunity inducing activity according to any one of claims 9 to 20 (claim 20), and claims 15 to 20.
An immunity-inducing activity promoting substance obtained by the screening method according to any one of the above (Claim 21) and Claims 15 to 2.
A immunity-inducing activity-suppressing substance obtained by the screening method of any one of claims 0 to 22 (claim 22).

Further, the present invention provides any one of claims 4 to 6,
Alternatively, an antitumor agent (Claim 23) containing the protein or peptide according to Claim 10 or the immunity-inducing activity promoting substance according to Claim 21 as an active ingredient (Claim 23), or Claims 4 to 6
Or an activated T cell induced by in vitro stimulation with the protein or peptide according to claim 10 or the immunity-inducing activity promoting substance according to claim 21 (claim 24); A DNA encoding any of the above or the protein or peptide according to claim 10.
Bladder cancer, brain tumor, malignant melanoma, chronic myelogenous leukemia, acute myeloid leukemia, lymphoma, esophageal cancer, kidney cancer, prostate cancer, consisting of all or part of the antisense strand of A or RNA,
Probe for diagnosis of lung cancer, pancreatic cancer, breast cancer, colon cancer, gastric cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, sarcoma (claim 2)
5) a bladder cancer, a brain tumor, a malignant melanoma, a chronic myelogenous leukemia, an acute myeloid, which comprises the diagnostic probe for cancer according to claim 25 and / or the antibody according to claim 8 or 9; Diagnostic agent for leukemia, lymphoma, esophageal cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, breast cancer, colon cancer, stomach cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, sarcoma (Claim 2)
6).

In the present invention, cDNA is prepared using mRNA obtained from a bladder cancer cell line, and
A bladder cancer cell-derived cDNA library prepared by infecting Escherichia coli by introducing it into a phage vector, a bladder cancer antigen derived from a bladder cancer cell obtained by screening a λ phage cDNA clone using a bladder cancer patient serum. A cDNA is prepared using an antitumor agent (claim 27) characterized in that the mRNA is obtained from a bladder cancer cell line, and the cDNA is introduced into a λ phage vector to infect E. coli. The prepared bladder cancer cell cDNA library, λ phage cDN
A clone obtained by screening using a serum of a bladder cancer patient, the whole or a part of the antisense strand of DNA or RNA encoding a bladder cancer antigen derived from a bladder cancer cell, or the bladder derived from the bladder cancer cell The present invention relates to a diagnostic agent for detecting a tumor, which comprises an antibody against a cancer antigen (claim 28).

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The target protein of the present invention includes a bladder cancer antigen KU shown in SEQ ID NO: 2 in the sequence listing.
-BL-50 or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and which has an immunity-inducing activity, Here, the immunity-inducing activity refers to an activity of inducing an immune reaction such as antibody production, cell-mediated immunity, or immune tolerance. Among such immunity-inducing activities, the frequency of peripheral blood cytotoxic T cell (CTL) precursor cells Those having a T cell-inducing activity for increasing the T cell are particularly preferred. Further, as the peptide of the present invention,
It is not particularly limited as long as it is a peptide consisting of a part of the above protein and having immunity inducing activity,
Antibody recognition sites, CD4 + T cells and / or CD8 +
Peptides constituting the recognition site of T cells are preferred. The above-mentioned proteins and peptides of the present invention, and recombinant proteins and peptides to which antibodies specifically binding to these proteins and peptides specifically bind, may be collectively referred to as “the bladder cancer antigen” below. . The origin of the bladder cancer antigen is not limited to humans.

The DNA to be used in the present invention includes a protein KU consisting of the amino acid sequence shown in SEQ ID NO: 2.
-A DNA encoding BL-50, which comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2,
Encoding a protein having immunity-inducing activity
A, the base sequence shown in SEQ ID NO: 1 or its complementary sequence, and a DNA containing a part or all of these sequences can be exemplified. Such a bladder cancer antigen KU-BL-50
The method for preparing the cDNA encoding is not particularly limited. For example, the cDNA may be prepared by introducing a cDNA prepared using mRNA obtained from a bladder cancer cell line into a λ phage vector and infecting E. coli. The bladder cancer cell cDNA library screened using the bladder cancer patient serum, reacting only with the bladder cancer patient serum, not reacting with healthy human serum, and expressing the bladder cancer antigen expressed in cancer cells including bladder cancer Can be obtained by screening.

Further, using the nucleotide sequence shown in SEQ ID NO: 1 or a sequence complementary thereto and a part or all of these sequences as a probe, hybridization is carried out under stringent conditions with DNA libraries derived from various bladder cancers. And the DNA hybridizing to the probe
Can also be obtained to obtain a DNA encoding a protein having a desired immunity-inducing activity, which is the same as that of the bladder cancer antigen KU-BL-50. The DNA thus obtained is also within the scope of the present invention. Such DNA of the present invention
Examples of the hybridization conditions for obtaining a compound include hybridization at 42 ° C. and washing at 42 ° C. with a washing buffer containing 1 × SSC and 0.1% SDS. Hybridization at 0.1 ° C. and 0.1 × SSC, 0.
Washing at 65 ° C. using a washing buffer containing 1% SDS can be more preferably mentioned. In addition, as factors that affect the stringency of hybridization, there are various factors other than the above-mentioned temperature conditions, and those skilled in the art can appropriately combine various components,
It is possible to realize a stringency equivalent to the stringency of hybridization exemplified above.

The fusion protein or fusion peptide of the present invention may be any fusion protein or marker protein in which the bladder cancer antigen is linked to a marker protein and / or a peptide tag. The marker protein is not particularly limited as long as it is a marker protein, and specific examples thereof include alkaline phosphatase, an Fc region of an antibody, HRP, and GFP.
Conventionally known peptide tags such as His tag, FLAG tag, and S tag can be specifically exemplified. Such a fusion protein can be prepared by a conventional method, and purifies a bladder cancer antigen KU-BL-50 or the like using affinity of Ni-NTA and a His tag, detects a protein having T cell inducing activity, Bladder cancer antigen KU-BL-5
It is useful as a quantification of an antibody against 0, etc., as a diagnostic marker for bladder cancer, and as a research reagent in the field.

Specific examples of the antibody that specifically binds to the protein or peptide of the present invention include immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, and humanized antibodies. These can be prepared by a conventional method using a protein such as the above-mentioned bladder cancer antigen KU-BL-50 or a part thereof as an antigen, and among them, a monoclonal antibody is preferable in terms of its specificity. A monoclonal antibody that specifically recognizes the KU-BL-50 epitope or a complex of the epitope and HLA is more preferable. Antibodies such as monoclonal antibodies, for example, diagnosis of bladder cancer and the like,
It is useful in elucidating the onset mechanism of malignant tumors such as bladder cancer as well as treatments such as missile therapy.

Further, the antibody of the present invention can be obtained by using a conventional protocol in an animal (preferably a non-human animal) or a fragment thereof containing the bladder cancer antigen or an epitope of the bladder cancer antigen, particularly the epitope and HLA, in an animal (preferably other than human). The hybridoma method (Na) is produced by administering cells that express the complex on the membrane surface, for example, for the preparation of monoclonal antibodies, which results in antibodies produced by continuous cell line cultures.
Nature 256, 495-497, 1975), Trioma method, human B cell hybridoma method (Immunology Today 4, 72, 1983)
And EBV-hybridoma method (MONOCLONAL ANTIBODIE
S AND CANCER THERAPY, pp.77-96, Alan R.Liss, Inc.,
1985).

In order to prepare a single-chain antibody against the bladder cancer antigen of the present invention, a method for preparing a single-chain antibody (US Pat. No. 4,946,778) can be applied. Further, in order to express a humanized antibody, using a transgenic mouse or other mammals or the like, using the above antibody,
The clone expressing the bladder cancer antigen of the present invention can be isolated and identified, or the polypeptide can be purified by affinity chromatography. The antibody against the bladder cancer antigen or a peptide containing the antigen epitope may be used for diagnosis or treatment of bladder cancer or the like. Recombinant proteins or peptides to which these antibodies specifically bind are also included in the bladder cancer antigen of the present invention as described above.

The present invention also relates to a host cell comprising an expression system capable of expressing the bladder cancer antigen and an H system comprising an expression system capable of expressing the bladder cancer antigen.
The present invention relates to a host cell capable of expressing LA. Introduction of such a gene encoding the bladder cancer antigen into host cells is carried out by Davi
(BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANU
AL, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989) and methods described in many standard laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection,
ransvection), microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading
ng), ballistic introduction, infection, etc.

The host cells include Escherichia coli,
Bacterial prokaryotic cells such as Streptomyces, Bacillus subtilis, Streptococcus, and Staphylococcus, yeast, fungal cells such as Aspergillus, insect cells such as Drosophila S2 and Spodoptera Sf9, L cells, CHO cells, COS cells, HeLa cells, C127 cells, BALB / c3T3
Cells (including mutants lacking dihydrofolate reductase, thymidine kinase, etc.), BHK21 cells, HEK
293 cells, Bowes bladder cancer cells and other animal cells, plant cells and the like. Examples of the host cells having the ability to express HLA include not only cells having the ability to express HLA but also cells having the ability to not express HLA.
Examples include cells transfected with cDNA.

The expression system may be any expression system capable of expressing the bladder cancer antigen of the present invention in a host cell, and expression systems derived from chromosomes, episomes and viruses, for example, From bacterial plasmids, yeast plasmids, papovaviruses such as SV40, vaccinia virus, adenovirus, adenoassociated virus, fowlpox virus, pseudorabies virus, retrovirus-derived vectors, bacteriophage-derived, transposon-derived and combinations thereof. Derived vectors include, for example, those derived from plasmids such as cosmids and phagemids and genetic elements of bacteriophages. The expression system may include control sequences that regulate as well as cause expression.

The host cell comprising the above-mentioned expression system, the cell membrane of such a cell, and the bladder cancer antigen obtained by culturing such a cell can be used in the screening method of the present invention as described later. For example, as a method for obtaining a cell membrane, F. Pietri-Rouxel (Eur. J. Biochem., 247,
1174-1179, 1997).
Further, to recover and purify the bladder cancer antigen of the present invention from cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography,
Known methods including phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably high performance liquid chromatography, are used. In particular, as a column used for affinity chromatography, for example, a column to which an antibody against the bladder cancer antigen is bound, or when a normal peptide tag is added to the bladder cancer antigen, the column has affinity for the peptide tag. The bladder cancer antigen of the present invention can be obtained by using a column to which the substance is bound.

In the present invention, the above-mentioned non-human animal in which the function of the gene encoding the bladder cancer antigen is deficient on the chromosome is defined as a part or all of the gene encoding the bladder cancer antigen on the chromosome which is disrupted or deleted. A non-human animal that has been inactivated by a gene mutation such as substitution and has lost the function of expressing the bladder cancer antigen, and a non-human animal that overexpresses the bladder cancer antigen is a wild-type non-human animal Refers to non-human animals that produce such bladder cancer antigens in large amounts.
And, as the non-human animal in the present invention, a mouse,
Specific examples include non-human animals such as rodents such as rats, but are not limited thereto.

By the way, the homozygous non-human animals born according to Mendel's law include the bladder cancer antigen-deficient or overexpressed type and the wild type of the homozygous non-human animal. The simultaneous use of the deficient or overexpressed type and its littermate wild type enables accurate comparison experiments at the individual level, so that the function of the gene encoding the wild type non-human animal, namely the bladder cancer antigen, is It is preferable to use an animal of the same species as the non-human animal deficient or overexpressed on the chromosome, or an animal of the same litter, in the screening of the present invention described below, for example. The method for producing such a non-human animal in which the gene function encoding the bladder cancer antigen is deficient or overexpressed on the chromosome will be described below using a knockout mouse or a transgenic mouse of the bladder cancer antigen as an example.

For example, a mouse in which the gene function encoding the bladder cancer antigen is deficient on the chromosome, that is, a bladder cancer antigen knockout mouse is obtained by using a gene fragment obtained from a mouse gene library by a method such as PCR. The gene encoding the subject bladder cancer antigen is screened, the screened gene encoding the bladder cancer antigen is subcloned using a viral vector or the like, and identified by DNA sequencing. All or part of the gene encoding the bladder cancer antigen of this clone is replaced with a pMC1 neo gene cassette or the like, and the diphtheria toxin A fragment (DT-A) gene or the herpes simplex virus thymidine kinase (HSV) is
-Tk) A targeting vector is prepared by introducing a gene such as a gene.

The thus prepared targeting vector is linearized, introduced into ES cells by electroporation (electroporation) or the like, and subjected to homologous recombination. Among the homologous recombinants, G418 or ganciclovir is obtained. (G
E.A. which has undergone homologous recombination with an antibiotic such as
Select S cells. In addition, it is preferable to confirm whether or not the selected ES cell is the desired recombinant by Southern blotting or the like. The confirmed ES cell clone is microinjected into a mouse blastocyst, and the blastocyst is returned to a foster parent mouse to produce a chimeric mouse. When this chimeric mouse is intercrossed with a wild-type mouse, a heterozygous mouse can be obtained, and the bladder cancer antigen knockout mouse of the present invention of the present invention is produced by intercrossing the heterozygous mouse. be able to. In addition, as a method for confirming whether or not the bladder cancer antigen knockout mouse has occurred, for example, RNA is isolated from the mouse obtained by the above method and examined by Northern blotting or the like, or expression of this mouse Is examined by Western blotting or the like.

The transgenic mouse of the bladder cancer antigen was prepared by adding chicken β to the cDNA encoding the bladder cancer antigen.
-Actin, mouse neurofilament, promoters such as SV40, and rabbit β-globin, SV40
A transgene is constructed by fusing such poly A or intron, and the transgene is microinjected into the pronucleus of a mouse fertilized egg, and the obtained oocytes are cultured, and then transplanted into the oviduct of a foster parent mouse. Such a transgenic mouse can be created by breeding a recipient animal and selecting a pup mouse having the above cDNA from the pup mice born. In addition, selection of offspring mice having cDNA can be carried out by extracting a crude DNA from the tail or the like of the mouse and conducting a dot hybridization method using a gene encoding the bladder cancer antigen introduced as a probe or a PCR method using specific primers. And so on.

Further, the gene or DNA encoding the bladder cancer antigen, the bladder cancer antigen, the fusion peptide or fusion protein in which the bladder cancer antigen is linked to a marker protein and / or a peptide tag, and the bladder cancer antigen Host cells and the like comprising the antibody and the expression system capable of expressing the bladder cancer antigen of the present invention include brain tumors including vesical cancer (glioma) and malignant melanoma (melanoma), as specifically described below. , Chronic myeloid leukemia,
Acute myeloid leukemia, lymphoma, esophageal cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, breast cancer, colon cancer, stomach cancer, liver cancer, gallbladder cancer,
It is useful in the treatment and diagnosis of testicular cancer, uterine cancer, ovarian cancer, sarcoma, etc., and is used for screening for substances that promote or suppress immunity-inducing activity, for detecting activated T cells or their precursor cells, and for reagents for such detection. Not only can be used, but also activated T cells (CD4 antigen positive T cells, CD8 antigen positive T cells)
Cells, helper T cells, killer T cells, suppressor T
And the elucidation of the mechanism of an immune response such as induction of all T cells (including cells).

As a method for screening a substance for promoting or suppressing immunity-inducing activity of the present invention, a method for measuring and evaluating immunity-inducing activity on T cells using a test substance, the bladder cancer antigen and T cells, A method for measuring and evaluating immunity-inducing activity in T cells using a test substance, a cell membrane or cells expressing the bladder cancer antigen and T cells, and HLA
A method for measuring and evaluating immunity-inducing activity in T cells by using a bladder cancer antigen-expressing host cell transfected with a vector expressing a test polypeptide and a vector expressing a test polypeptide; Using a bladder cancer antigen-expressing host cell capable of expressing HLA transfected with a peptide-expressing vector and a T cell,
A method for measuring and evaluating the immunity-inducing activity in cells, a method for administering a test substance to a non-human animal such as the knockout mouse and the transgenic mouse, and measuring and evaluating the immunity-inducing activity in T cells of the non-human animal, etc. Can be mentioned. Examples of the cell membrane or cells include cells such as primary cultured cells obtained from a non-human animal or a wild-type non-human animal in which the gene encoding the bladder cancer antigen is overexpressed, and the bladder cancer antigen of the present invention. Specific examples include host cells comprising an expression system capable of expression, cell membranes of these cells, and the like. Examples of the method of contacting such a cell membrane or cells with a test substance include the presence of the test substance. A method of culturing the cell membrane or cells expressing the bladder cancer antigen in vitro in vitro and then contacting the cells with T cells can be exemplified. As a method of measuring / evaluating the immunity-inducing activity in T cells, a method of evaluating using the amount of IFNγ released from T cells into the medium as an index can be specifically exemplified.

The present invention also provides a substance for promoting immunity-inducing activity, which is used for the treatment of a patient in need of promoting the immunity-inducing activity obtained by the above screening method, and a method for treating a patient in need of suppressing the immunity-inducing activity. The present invention relates to a substance for suppressing an immunity-inducing activity, which is used for: The present invention also provides a bladder cancer containing the bladder cancer antigen as an active ingredient, a brain tumor,
Malignant melanoma, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, esophageal cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, breast cancer, colon cancer, gastric cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, It relates to an antitumor agent for sarcoma. For example, when the bladder cancer antigen is administered orally, intravenously, intradermally, subcutaneously, or the like, an antitumor effect due to an increase in T cell inducing activity in vivo can be expected. Further, the above antibody can be used for missile therapy. The present invention also relates to activated T cells induced by in vitro stimulation with the bladder cancer antigen. For example, stimulation of peripheral blood lymphocytes or tumor-infiltrating lymphocytes with the bladder cancer antigen together with IL-2 induces tumor-reactive activated T cells, and the activated T cells can be used effectively for adoptive immunotherapy. Can be. Further, the bladder cancer antigen of the present invention can be expressed in vivo or in vitro in dendritic cells which are powerful antigen-presenting cells, and immunity can be induced by administering the antigen-expressing dendritic cells.

Further, the present invention provides a diagnostic probe for cancer comprising all or a part of the antisense strand of DNA or RNA encoding the bladder cancer antigen, a diagnostic probe for the cancer and a specific probe for the bladder cancer antigen. Bladder cancer, brain tumor, malignant melanoma, chronic myelogenous leukemia, acute myelogenous leukemia, lymphoma, esophagus cancer, kidney cancer, prostate cancer,
The present invention relates to a diagnostic agent for lung cancer, pancreatic cancer, breast cancer, colon cancer, gastric cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, sarcoma and the like. Examples of the diagnostic probe include D which encodes the bladder cancer antigen of the present invention.
Preferably, the antisense strand is a whole or a part of the antisense strand of NA (cDNA) or RNA (cRNA) and has a length (at least 20 bases or more) that can be established as a probe. By detecting the mRNA of the bladder cancer antigen obtained from the specimen, bladder cancer, brain tumor, malignant melanoma, chronic myelogenous leukemia, acute myeloid leukemia, lymphoma, esophageal cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer It is possible to diagnose diseases such as breast cancer, colon cancer, gastric cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, and sarcoma. Specific examples of the sample used for such detection include, but are not limited to, genomic DNA, RNA, and cDNA that can be obtained from a biopsy of a subject's cells, such as blood, urine, saliva, and tissue. Instead, when such a sample is used, a sample amplified by PCR or the like may be used.

The present invention also provides a method for preparing cDNA using mRNA obtained from a bladder cancer cell line,
A bladder cancer cell-derived cDNA library obtained by infecting Escherichia coli by introducing NA into a λ phage vector, a bladder cancer cell-derived bladder obtained by screening a λ phage cDNA clone using a bladder cancer patient serum An antitumor agent characterized by containing a cancer antigen, or all or a part of the antisense strand of DNA or RNA encoding the bladder cancer antigen derived from the bladder cancer cell, or an antibody thereto. The diagnostic agent for tumor detection described above is also included.

[0037]

EXAMPLES The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Preparation of bladder cancer cell line] KU-19-19 was established from perineal metastasis of grade 3 transitional cell carcinoma, KU-7 was established from grade 1 transitional cell carcinoma, and KU-1 was established from grade 2 transitional cell carcinoma. These three established bladder cancer cell lines were identified as RPMI1
640 (10% fetal calf serum, 100 IU / ml penicillin, 100 μg / ml streptomycin, 2
mM L-glutamine, 10 mM HEPES)
At 37 ° C., 5% CO ₂ and 100% humidity. After collecting a sufficient amount of cells, freeze in liquid nitrogen-
Stored at 80 ° C.

[Serum treatment for cDNA cloning] As a serum used for cDNA screening, a serum of a 71-year-old patient having an invasive bladder tumor with metastasis was used. In addition, histopathological diagnosis of the tumor of this patient showed a histological image of grade 3 transitional cell carcinoma, partially differentiated into squamous cell carcinoma, adenocarcinoma, and sarcomatous carcinoma. The local progression of the tumor was found to involve the deep muscular layer and was accompanied by lymphatic invasion. The serum after blood collection was stored at -80 ° C. To adsorb the antibody against Escherichia coli in the serum, 1 ml of the serum was mixed with a lysate of Escherichia coli (XL1-Blue MRF 'stain) at a ratio of 1: 5, left at 4 ° C. for 8 hours, and then left at 150 ° C.
After centrifugation at 00xg for 20 minutes, the supernatant was recovered. After diluting the supernatant 100-fold with a mixed solution of TBS (Tris-buffered saline) and 5% skim milk powder (wt / vol.),
Furthermore, in order to absorb the antibody against the vector system, a protein derived from Escherichia coli and phage was adsorbed (protein produced from λ phage without an insert was transferred), and a 15 cm nitrocellulose filter (Hybond-C, Amersham Pharmasia biot
(ech, Buckinghamshire, England) Twenty pieces (per 1 ml of serum) were put in the diluted serum and shaken at room temperature for 8 hours to adsorb non-specific anti-E. coli and anti-phage antibodies in the supernatant. Used after processing.

[Lambda phage cDNA from bladder cancer cell line
Preparation of Library] Three Cultured Bladder Cancer Cell Lines
(Each 1 × 10⁸) To guanidine-cesium chloride
RNA is extracted by the heart method, and mRNA is converted to oligo (dT) Olig
otex-dT30 super mRNA Purification Kit (Takara Shuz
ou, Kyoto, Japan) using the poly (A) selection method.
NA was purified. Poly (A) from three purified cell lines
RNA was mixed, and 5 μg of the mixed total RNA was used for ZAP-cDNA.
 Using Synthesis Kit (Stratagene, La Jolla, CA)
Inserted into the λZAP Express vector, 2.0 × 10
⁶Bladder cancer lambda phage cDNA consisting of pfu clone
A library was made. Note that this cDNA library
Lee does not contain B lymphocytes, so IgG
CDNA clones to be loaded are not included.
No false positive clones are detected. This prepared cDN
A library is amplified and the following immunoscreening is performed.
It was used for.

[Immunoscreening of cDNA Library with Serum] The λ phage cDNA library prepared above was placed in a 15 cm NZY agarose plate at 0.
5-1 × 10 ⁴ cells were inoculated, cultured at 42 ° C. for 4 hours, and expressed on Escherichia coli (XL1-Blue). IPT on this agar medium
Put a nitrocellulose filter that has absorbed G
After culturing at 7 ° C for 4 hours, the expressed fusion protein was transferred to a nitrocellulose filter, and TBST was added.
(10 mM Tris-HCl, 150 mM NaC
After washing the filter with 0.5% Tween 20 (pH 7.4), the filter was allowed to stand in TBS / 5% non-fat dry milk for about 8 hours to suppress nonspecific reaction. The filter and the serum subjected to the adsorption treatment were reacted at room temperature for 4 hours, and the plaque in which the antibody in the serum reacted was converted into a 4000-fold diluted anti-human IgG-Fc enzyme-labeled antibody (antihum).
an IgG Fc goat antibody alkaline phophate conjugat
ed, CAPPEL) and react with nitro blue tetrazoli
um (Roche) and 5-bromo-4-chloro-3-indolyl (Sigma-Ald
rich Japan, Tokyo, Japan).
cmNZY agarose plate, and collected in SM buffer (100 mM NaCl, 10 mM MgSO ₄ , 5
0 mM Tris-HCl, 0.01% gelatin; p
H7.5). The secondary and tertiary screening was repeated in the same manner as described above until the chromogenic reaction-positive plaque was unified, and a single phage clone to which the IgG antibody in the serum reacted was obtained. Using patient serum, SER
Immunoscreening by X method (total of 4.0 × 10
Screening ⁵ lambda phage clones)
39 positive clones were isolated.

[Search for Homology of Isolated Antigen Gene] From the 38 phages obtained above, PCR (polymerase chain) was performed.
The insert DNA was amplified by the reaction) method and used for the subsequent analysis. Ex-Taq (Takara S
huzo) and T3 (5'-AAT) as a sense primer.
TAA CCC TCA CTA AAG GG-
3 ': SEQ ID NO: 3), T7 as an antisense primer
(5'-GTA ATA CGA CTC ACAT
AGGGC-3 ': SEQ ID NO: 4) was used. The reaction conditions were thermal denaturation using a thermal cycler (Perkin-Elmer) at 94 ° C. for 1 minute, annealing at 55 ° C. for 2 minutes,
35 cycles of the extension reaction at 72 ° C. for 2 minutes were performed. The obtained PCR product is used for Big Dye DNA Sequencing.
DNA sequencing was performed using a kit (PE Biosysems, CA) and an ABI310 autosequencer (PE Biosysems, CA), and a base sequence of 300 to 650 bp was determined first. As a result, 28 different genes were obtained.

The nucleotide sequences of the 28 types of genes determined above were respectively entered into the homology search program BLAST (Basi
c Using the Local Alignment Search Tool)
This was compared with the genetic information registered in the data bank.
As a result of the search in the database, if the molecule is a known molecule, a molecule associated with cancer in the past literature is selected as a cancer antigen candidate gene, and if the molecule is unknown, a DNA matching in the DNA database is selected. Cluster [expression seq
uence tag (EST) is limited to cancer tissues, cancer cell lines, fetal tissues, limited normal tissues such as testis, etc., and specific expression patterns are expected. Was selected as a candidate. The results are shown in Table 1 (17 types of known antigen genes) and Table 2 (11 types of unknown antigen genes). In addition, the unknown gene is EST (expresse
d sequence tags), KIAA (KIAA proteins; KAZUS
A, Chiba, Japan) and DNA database (GenBank)
etc.) registered on the DNA fragment sequence (DNA clust
ers). Also,
7 for KU-BL-76 / pinch protein, KU-B
L-49 / nucleophosmin homolog, KU-BL-50
/ Ecalectin homolog, KU-BL-52 / EST, K
2 for U-BL-55 / liprin beta-1
Duplicate clones were detected.

[0043]

[Table 1]

[0044]

[Table 2]

It was possible that 7 of 28 antigen genes were associated with cancer. KU-BL-
49 is 94.7% at the nucleotide sequence level with nucleophosmin,
There is 98.3% homology at the amino acid sequence level, and from the amino acid sequence, it is highly possible that the gene is a nucleophosmin family gene. Nucleophosmin has many reports related to cancer. For example, in anaplastic large cell lymphoma, nucleophosmin has been reported to fuse with the ALK gene and cause malignant transformation. In breast cancer, nucl
Antibody titers to eophosmin have been reported to predict recurrence of breast cancer. If KU-BL-49 also has a similar function, it is highly likely that it is a molecule associated with cancer. KU-
BL-50 is also 75.6% at the nucleotide sequence level with galectin family ecalectin, and 7 at the amino acid sequence level.
There was 9.2% homology. Ecalectin is an eosinophil attractant (chemoattractan) secreted from T lymphocytes.
t). Recently, the galectin family has been reported to be associated with cancer. For example, Galectin
-9 has been reported to be highly expressed in Hodgikin 'disease. Galectin-4, which was isolated by the SEREX method for colorectal cancer, shows an expression pattern restricted to the gastrointestinal tract and is one of the colorectal cancer antigen candidates. KU-BL
Given that -50 is a member of the galectin family, these galectins may be associated with cancer.

The KU-BL-67 / nmb gene was proved to have a reduced metastatic ability upon gene transfer in a melanoma cell line, and was a negative regulator of biological activities.
It is suggested that KU-BL-68 / plas
minogen activator inhibitor-1 is used for tumor metastasis, invasion,
It is thought to be involved in migration. KU-BL-72 (= KU-BL-1), KU-B
L-73 (= KU-BL-3) is a unique group of tissues derived from the corresponding DNA cluster and is an important antigen. Has been isolated. Also K
Although U-BL-75 / LBc protooncogene is not oncogene, it is possible that IgG antibodies produced in response to oncogene were detected by cross-reaction. LBc protoonc
ogene is located on chromosome 5 but onco-LBc is LBc proto
It has been reported that the N-terminus of -oncogene is activated by replacing the short C-terminus from chromosome 7.

Also, considering the tissues from which the DNA clusters that correspond to other unknown genes are identical, there were some molecules whose specific expression patterns were expected.
KU-BL-51 is EST (H62686, Unigene number: 173).
094). Many matching ESTs
Was from fetal tissue (eg fetal liver and spleen, fetus, etc.). KU-BL-52 is EST (AW97741)
2) but it was not registered with UniGene. ESTs that almost match are germinal center B cells,
Fetal and neonatal brain tissue, carcinoids, etc.
KU-BL-53 was almost identical to EST (AW514723, Hs. 183861). The ESTs with almost the same origin were cancer tissues (eg, endometrial cancer) and normal prostate.

[Detection of IgG Antibody Against Each Isolated Cancer Antigen in Serum of Normal Person and Serum of Cancer Patient] The phage clone obtained above and a negative control phage containing no insert (a phage containing no cDNA insert) Were mixed so as to express at a ratio of 1: 1, and a total of 1 × was placed on a 15 cm NZY agarose plate.
10 ⁴ seeded (isolated clones 5 × 10 ³ cells and the control phages 5 × 10 ³ cells) of the clones after transfer protein to 15cm nitrocellulose membrane, the following screening by cutting the membrane to ten It was used for. The sera of healthy subjects and bladder cancer patients were screened by diluting them 100-fold with a mixture of TBS and 5% skim milk powder.

From the literature review and the tissue from which the matching DNA clusters were derived, 24 of 28 antigen genes were selected as bladder cancer antigen candidate genes. In the serum reaction, whether or not the antibody was present in the serum of a healthy individual was first examined by the above-mentioned screening. Serum from healthy volunteers was healthy at the time of blood collection, and blood was collected from 16 volunteers (9 males and 7 females) with an average age of 27.8 years without a history of autoimmune disease, as described above. Adsorbed serum was used. As a result, 24
Three of the clones were found to react with antibodies in the sera of multiple healthy individuals. These clones were excluded from further consideration. The presence of antibodies in healthy human sera does not necessarily rule out the association with cancer, but in this study clones that reacted with multiple healthy human sera were excluded from further studies. However, healthy human serum 1
A clone (KU-BL-
55 / liprin beta-1) is important as in NY-CO-38 if the antibody is present in the serum of the donor at a low rate even if the antibody is present in the serum of a healthy individual. May contain antigens (Int. J. Cancer 76,
652-8, 1998).

The presence of antibodies in the sera of 27 bladder cancer patients other than the above-mentioned patients was examined in the same manner as described above for 21 clones screened using healthy human serum. Table 3 shows the results of 14 clones among the results of examining the presence of IgG antibodies. 7 out of 21 clones
The clone reacted with the antibody in the serum of a bladder cancer patient other than the patient used in the above screening, and the remaining 1
Four clones reacted only with the antibody in the serum of patient A used for the primary screening. KU-BL-73 (8 clones), in which the most clones were detected,
It was considered that some degree of immune reaction occurred in bladder cancer patients. Moreover, KU-BL-50 is also 2
Three antibodies were found in the sera of eight bladder cancer patients (including the patient sera). In addition, KU-BL-55 / liprin
-beta-1, KU-BL-56 / RasGAP-related protei
n, KU-BL-62 / SFRS protein kinase 1, KU-
BL-74 / high density lipoprotein binding protein
In clones such as in, KU-BL-72 / EST (= KU-BL-1) and the like, IgG antibodies were detected in the sera of bladder cancer patients other than the above patients.

[0051]

[Table 3]

Also, 10 cases of melanoma, 10 cases of brain tumor,
Serum from 10 cases of pancreatic cancer, 10 cases of esophageal cancer, 10 cases of renal cell carcinoma, 14 cases of prostate cancer, 7 cases of colorectal cancer, and 6 cases of testicular tumor was used. Examination of the presence or absence of IgG antibodies (Table 3) revealed that few clones had antibodies in the serum of cancer patients other than bladder cancer. Among them, KU-BL-50
/ Ecalectin homolog, KU-BL-55 / liprin-bata
-1, KU-BL-75 / Lbc protooncogene and the like confirmed the presence of IgG antibodies in the serum of cancer patients other than bladder cancer. In particular, KU-BL-50 was found to be present in antibodies in the sera of several cancer patients. KU-BL-50 showed an antibody in the serum of 3 of 28 bladder cancer patients, and 6 of 10 esophageal cancer patients,
Antibodies were also present in the serum of 5 of 14 prostate cancer patients, 3 of 10 renal cancer patients, 1 of 10 pancreatic cancer patients, and were found in esophageal cancer, renal cell carcinoma, and prostate cancer. Since IgG antibody was present at a rate of 30% or more in each case, KU-BL-50 was considered to be an antigen having immunogenicity not only in bladder cancer but also in other cancers. Also, KU-BL-75 / Lbc protooncogene
Is not an oncogene, but the IgG antibody produced in response to the oncogene may have been detected due to a cross-reaction.

[RT-PCR Method for Detecting mRNA Expression of Isolated Cancer Antigen] A relatively high frequency (1
The expression specificity of each of the 24 antigen genes in each tissue excluding the antigens in which the antibody was detected in 2 or more of 6 cases) was determined by RT-PC.
I examined it by the R method. A panel of cDNA was prepared using RNA extracted from normal tissues, cultured proliferating cells, and cultured cancer cell lines, and RT-PCR was performed. Brain, heart, kidney,
Total RNA from normal tissues such as spleen, liver, small intestine, smooth muscle, lung, testis, placenta, stomach, and large intestine was obtained from Clontech (Palo A).
lto, CA). Normal bladder tissue was purchased from Invitrogen (Carlsbad, CA). Bladder cancer cell lines (FY, KU1, KU7, KU19-19, NBT, T
24), proliferating cells (cultured fibroblasts, tumor infiltrating T cells;
TIL1364, EB virus infected B cells; 1088E
BV-B), cultured cancer cell lines other than bladder cancer [brain tumor (U8
7-Mo), melanoma (SKmel23), chronic myeloid leukemia (K-562), acute myeloid leukemia (HL-6)
0), esophageal cancer (TE8), renal cell carcinoma (KU-19-2)
0), prostate cancer (PC3), lung cancer (K1S), pancreatic cancer (P
K1)] was cultivated to reach 0.5 to 1.0 × 10 ⁸ , and total RNA was extracted by guanidine-cesium chloride ultracentrifugation.

Using 5 μg of each of the above total RNAs, Super Script II reverse transcriptase (GIBCO
/ BRL, Life Technologies, Inc., Rockville, MD, US
A) and a panel of cDNAs were prepared using oligo (dT) primers. From the nucleotide sequence of the clone obtained in the primary screening, specific primers are designed so that a PCR product of 300 to 700 bp can be produced, and Ex.
-Using Taq (Takara), heat denaturation at 94 ° C for 1 minute,
Annealing was performed for 25 cycles of PCR under the conditions that the optimal temperature of each primer was 2 minutes and the extension reaction was 72 ° C. for 2 minutes. The annealing temperature was set in consideration of the optimal Tm value of the primer.

KU-BL-50 had a sense primer (5'-CTC ATG TCCAGT GGA AG
CAG-3 ': SEQ ID NO: 5) and an antisense primer (5'-ACC ACC GCG TTC TCC
TTG AA-3 ′: SEQ ID NO: 6) was prepared and 626b
The p band was detected. Annealing temperature 57.5 ° C
And The control β-actin contained a sense primer (5′-ATCTGG CAC CAC ACC ACC).
TTC TAC AAT GAG CTGCG-3 ':
SEQ ID NO: 7), an antisense primer (5′-CG
T CAT ACT CCT GCT TGC TGA
TCC ACA TCT GC-3 ': SEQ ID NO: 8)
And heat-denatured at 94 ° C. for 30 seconds for annealing 68.
25 cycles of PCR were performed at 2 ° C. for 2 minutes and extension reaction at 72 ° C. for 2 minutes, and an 838 bp band was detected. KU-BL-49 has a sense primer (5'-
GCC ACC AAC CCA TGG AAG A
T-3 ': SEQ ID NO: 9) and an antisense primer (5'-GGC TGG AGT ATC CCG T
AT TG-3 ': SEQ ID NO: 10) was replaced with KU-BL-5
1 contains a sense primer (5'-ATA CAG C
TG GGG AGG TAG CT-3 ': SEQ ID NO: 11) and an antisense primer (5'-GGA G
AA GCT TGA GAT GCT CC-3 ':
SEQ ID NO: 12) and KU-BL-52 were added to a sense primer (5'-CCC GCG CCG ATG TG
AATT ATT-3 ': SEQ ID NO: 13) and an antisense primer (5'-CCG CAT CTC CA
G GGT CTT TAA-3 ': SEQ ID NO: 14)
KU-BL-53 has a sense primer (5'-
AAG GAG CTG CTA CAG CCA C
A-3 ': SEQ ID NO: 15) and an antisense primer (5'-GCA CGC TGA AGC CAT A)
PCR was performed using AC AG-3 ': SEQ ID NO: 16).

The obtained PCR product was analyzed by 3% agarose gel electrophoresis, stained with ethidium bromide (EtBr), and the band was detected by irradiation with ultraviolet light at 254 nm. As a result, many isolated antigens were widely expressed in normal tissues. Among them, seven genes were expressed only in bladder cancer cell lines, other cancer cell lines, and limited normal tissues. These seven clones are KU-B
L-49 / nucleophosmin homolog, KU-BL-50
/ Ecalectin homolog, KU-BL-51 / EST, K
U-BL-52 / EST, KU-BL-53 / EST,
KU-BL-72 / EST (= KU-BL-1), KU
-BL-73 / EST (= KU-BL-3).
Of these, five clones (KU-BL-49,
KU-BL-50, KU-BL-51, KU-BL-5
2, KU-BL-53) are shown in Table 4. Also, R
Regarding the above-mentioned five types of clones in which a specific expression pattern was recognized by the T-PCR method, 30
PCR was performed for 35 cycles and 35 cycles, and RT-PCR was also performed on a panel of 23 bladder cancer tissues to examine expression. In addition, KU-BL-50 and KU-B
In L-52, RT-PCR panels of various cancers were expanded to show 11 cases of melanoma, 2 cases of glioma, and 6 cases of lung cancer.
Example, breast cancer 2 cases, renal cell carcinoma 11 cases, pancreatic cancer 6 cases, blood cell malignant tumor (leukemia and lymphoma) 4 cases, colorectal cancer 7 cases, esophageal cancer 1
Investigation was conducted in 6 cases, 3 cases of gastric cancer, and 3 cases of prostate cancer.

[0057]

[Table 4]

From the above results, KU-BL-50 was found to be expressed in bladder cancer, but in other normal tissues, normal proliferating cells and cancer cell lines, 25 and 35 cycles of RT-P were observed.
No expression was observed in CR. Also, KU-B
L-50 was expressed in two of six bladder cancer cell lines (KU-1 and N
BT) and expression in 13 of 23 bladder cancer tissues (Table 5). RT-PC for carcinomas other than bladder cancer
When the R panel was expanded and examined, expression was observed in limited carcinomas such as esophageal cancer, renal cell carcinoma, gastric cancer, and melanoma. In particular, the expression frequency in renal cell carcinoma was high. These results suggested that KU-BL-50 was expressed not only in bladder cancer but also in many other carcinomas.

[0059]

[Table 5]

KU-BL-49 / nucleophosmin homolo
In 25 cycles of RT-PCR, g was expressed only in testis in normal tissues, and weak expression was observed in many normal tissues in 30 cycles. Strong expression was observed in all six cases of bladder cancer cell lines. KU-BL-51 / E
ST was expressed in testis, lung, bladder, kidney, muscle, and brain in normal tissues in 25 cycles of RT-PCR. Among them, it was considered that the expression level was particularly high in testis. In proliferating cells, expression was observed in fibroblasts and in 2 out of 6 bladder cancer cell lines (FY, KU19-19). However, in 35 cycles, it was widely expressed in all normal tissues. In 25 cycles of RT-PCR, KU-BL-52 / EST normally showed expression in the brain and bladder. Among cancer cell lines, melanoma, esophageal cancer, brain tumor (glioma), and bladder cancer cell line (KU
1, KU7, NBT). Expression was also observed in some normal tissues in 35 cycles of RT-PCR. KU-BL-52 has 30 cycles of RT-
In PCR, expression was observed in 14 of 23 bladder cancer tissues. In addition, we expand panel of carcinoma except bladder cancer,
When expression was examined by RT-PCR, expression was observed in various carcinomas (Table 5).

[Northern Blotting Method for Detecting mRNA Expression of Isolated Cancer Antigen] Northern blotting was performed on the above five clones. 16 normal tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,
For thymus, prostate, testis, uterus, small intestine, large intestine, peripheral blood lymphocytes) panel, 2 polyA + RNAs from each tissue
Human multiple northern blots (Human Multiple Northern Blo
t) I and IV (Clontech) were used. Also,
Normal bladder, bladder cancer cell line (FY, KU1, KU7, KU
19-19, NBT, T24), for a panel of bladder cancer tissues, 20 total RNAs extracted from cell lines or tissues were used.
Those blotted to each lane by μg were used. These total RNAs were electrophoresed on an agarose gel containing formamide and formaldehyde, and were subjected to a nylon membrane (Hybond-N +, Amersham Pharmasia biotech, Tokyo, Ja.
pan).

Using the primers specific to the above five genes, a 500 to 1000 bp DN
A fragment (probe) is prepared, and this DNA fragment is
Labeled with ³² P using rimeDNA Labeling Kit (Boehringer). Using these labeled DNA fragments as probes, Salmon sperm using Quick Hyb (Stratagene, La Jolla, CA) as hybridization buffer
Prehybridization was performed with the DNA at 68 ° C. for 30 minutes. Then, add Quick Hy
Hybridized at 68 ° C for 2 hours in b. The membrane was washed with 2 × SSC, 0.1% SDS twice at room temperature for 15 minutes × 0.1 × SSC, 0.1% SDS.
This was performed once at 63 ° C. for 30 minutes using DS. Detection of radioactive signal is performed by Imaging Screen-K (35 × 43cm) au
toradiography films (Bio-RadLaboratories, Hercule
s, CA) and a Molecular Imager FX (Bio-Ra
d Laboratories, Hercules, CA). After removing the probe, human β-actin cDNA
Hybridization was performed in the same manner as above using a control probe (Clontech, Palo Alto, CA).

From the results of the above Northern blot, KU-
Similar to RT-PCR, BL-50 showed no expression in normal tissues, and 2 out of 6 bladder cancer cell lines (KU1 and NB)
Expression was observed in T). In addition, in about 12 representative bladder cancer tissues, a band of about 1.5 to 1.6 kbp was observed in 8 cases (FIG. 1). KU-BL-49 was also weakly expressed in normal tissues, and was slightly more strongly expressed in testis as in RT-PCR. It is strongly expressed in bladder cancer cell lines,
Strong expression was observed in cancer tissues in comparison between normal bladder tissues and bladder cancer tissues. KU-BL-51 was widely expressed in normal tissues, but the expression level in testis was very high. In comparison between normal bladder and bladder cancer tissue, strong expression was observed in the cancer tissue. KU-BL
-52 was expressed in the brain in normal tissues, and expressed in 2 out of 6 cases in the bladder cancer cell lines (KU1, NBT).
Weak expression was also observed in bladder cancer tissues.

[Comparison of mRNA Expression Levels by Real Time PCR] KU-BL-49 and KU-
BL-51 is a molecule suggested by Northern blot analysis to have a difference in expression level between normal bladder and bladder cancer tissue. The characteristics of these molecules include:
The expression level in testis is extremely high in normal tissues. This expression pattern is similar to the expression pattern of cancer-related molecules such as Her2 / neu, and KU-BL-49
And KU-BL-51 may also be molecules that are highly expressed in cancer. To prove high expression in cancer, real-time PCR was performed on normal bladder, other normal tissues, and bladder cancer tissues, and the expression levels of normal tissues and cancer tissues were examined. As the KU-BL-49 forward primer, 5'-CAA AGT GGA AGC CAA
GTT CAT C-3 '(SEQ ID NO: 17) was
-As a BL-49 reverse primer, 5'-CT
G CCA GAG ATC TTG AAT AGC
C-3 '(SEQ ID NO: 18) was used as a KU-BL-49 probe as 5'-(FAM) AAG AAT TG
T TTC CGG ATG ACT GAC CAG
G- (TAMRA) -3 'was used. KU-BL-5
As one forward primer, 5'-AAC C
AG AAG GCC AAG TGG AT-3 '
(SEQ ID NO: 19) was used as the KU-BL-51 reverse primer as 5′-CAG AAC TGT AGA
GCA AGC GCA G-3 '(SEQ ID NO: 20)
As a KU-BL-51 probe, 5 ′-(FA
M) TGC TGG CAA ACC CTG AAC
CTG TCT- (TAMRA) -3 'was used. Β-actin was used as a control. β-
As actin forward primer, 5'-TCA
CCC ACA CTG TGC CCATCT A
CG A-3 ′ (SEQ ID NO: 21) was used as a β-actin reverse primer as 5′-CAG CGG AA
C CGC TCA TTG CCA ATG G-
3 ′ (SEQ ID NO: 22) was used as a β-actin probe as 5 ′-(FAM) ATG CCC-X (TAMR
A) -CCC CCA TGCCAT CCT GCG
Tp-3 'was used. These primers and probes are available from Primer Express software (PE Biosystems, Fos
ter city, CA). Real-time PCR
ABI Prism 7700Sequ for amplification program and data analysis
This was performed using the ence Detector System (PE Biosystems).

1 μl of KU-BL-49 or KU-BL
-51 cDNA was mixed with 49 μl of master mix (Ta
gMan buffer, 3.5 mM MgCl ₂ , 300
nM of each of the above primers, 200 mM dATP, 20
0 mM dCTP, 200 mM dGTP, 200 mM
DUTP, 0.5U AmpErase Uracil N-glycosylas
e (UNG), 1.25U AmpliTaq Gold) plus PC
R was performed. PCR conditions were as follows: UNG treatment step at 50 ° C for 2 minutes, rTth DNA Polymerase reverse transcription reaction temperature and time setting at 60 ° C for 10 minutes, UNG
After inactivation at 95 ° C. for 10 minutes each,
PCR was performed for 60 cycles under the conditions that the primers were thermally denatured at 15 ° C. for 15 seconds and then annealed at the optimal temperature of each primer for 30 seconds. For each cDNA, two samples were subjected to PCR. In order to prepare a standard curve, PCR of each control cDNA (cDNA prepared from RNA of the bladder cancer cell line) was performed at five-step dilution, and simultaneously, PCR was performed. As a result, there was a tendency that the expression was higher in bladder cancer tissues than in normal bladder.

As described above, KU-BL-50 is not expressed in any of the normal tissues examined, is expressed only in cancer cells, and is recognized by the immune system when expressed in cancer cells. It is thought that it is. In addition to bladder cancer, it is mainly expressed in other epithelial cancers (renal cell carcinoma, esophagus cancer, gastric cancer), and it is highly expressed especially in renal cell carcinoma, and may be a common antigen for these cancers There is also.

[KU-BL-5 by 5'-RACE method]
0 Identification and Structural Analysis] KU-BL-50 isolated above
Since the ORF was not yet completed at the 5 'side, 5'-RACE (rapid amplification of cDNA ends) was used to isolate the full-length cDNA.
The method was performed. Marathon T as 5'-RACE kit
M cDNA amplification kit (Clontech, Palo Alto, CA,
USA) according to the instructions. Using 1 μg of poly (A) + RNA extracted from the bladder cancer cell line KU1 as a template, a first modification was carried out using a slightly modified oligo (dT) primer and the reverse transcriptase of avian myeloblastosis virus (AMV). Strand cDNA was synthesized.

For the synthesis of the second strand cDNA, Escherchia coli DNA polymerase I,
RNase H and DNA ligase were mixed and used. 2
The end of the single-stranded cDNA was blunt-ended with T4 DNA polymerase, and then Marathon using T4 DNA ligase.
cDNA adapter 1 (5'-CTA ATA CGACT
C ACT ATA GGG CTC GAG CGG
CCG CCCGGG CAG GT-3 ': SEQ ID NO: 23) was added to the 5' end to prepare a cDNA library. Using this library as a template, an adapter primer (AP1: 5'-CCA TCC
TAA TAC GAC TCACTA CTA TA
GGGC-3 ': SEQ ID NO: 24) and KU-BL-50
Primer GSP1 (5′-CCT CCC
GTT CAC TGTCAC CTG GAAC
PCR was performed using TG C-3 '(SEQ ID NO: 25). PCR conditions were 94 ° C. × 30 minutes, 94 ° C. × 5 seconds, 68 ° C. × 4 minutes, 5 cycles, 94 ° C. × 5 seconds, 7 cycles.
5 cycles of 0 ° C × 4 minutes, 94 ° C × 5 seconds, 72 ° C ×
25 cycles of 4 minutes were performed, and finally one cycle of 72 ° C. × 4 minutes was performed.

When 5 μl of the PCR product obtained above was electrophoresed in a 1% agarose gel and stained with ethidium bromide, a non-specific band was also detected. Therefore, the remaining PCR product was treated with a tricine-EDTA buffer (10 mM). Of Tricine, 0.1 mM EDTA) and diluted 50 times with template Primer 2 (AP2:
5'-ACT CAC TAT AGG GCT CG
A GCG GC-3 ': SEQ ID NO: 26) and KU-BL
-50-specific primer GSP2 (5'-TGT
TGC AGA CCA CGT AGC CGC C
CT CT-3 ′: nested PCR using SEQ ID NO: 27)
And a specific band was detected. KU-BL-50
A band which seems to be specific to the DNA was excised from a 1% agarose gel, the PCR product was purified, and then pGEM-T (Pr
TA cloning was performed using omega Inc., Madison, WI, USA). Perform colony PCR (Colony PCR)
A clone of the insert having the desired length was confirmed, and the nucleotide sequence of the insert was determined.

As a result, the length of the cDNA encoding KU-BL-50 was 1547 bp. This means that the length of the band by Northern blot is 1.5 to 1.6 kb.
p. Base sequence and OR of this cDNA
The amino acid sequence of F is shown in FIG. ORF is 46 to 103
Methionine which is 2 bp and starts at 64 bp is GXXA
He met TGT's Kozak rule. 43-45
A stop codon (TAA) was found at bp. KU-
BL-50 was considered to be a protein consisting of 323 amino acids. In homology search of base sequence,
The part of 46 'to 1002 bp on the 5' side is the urat of Sus scrofa.
It almost coincided with e transporter / channel protein (GenBank accession number: AJ131826). But urat
No information on e transporter / channel protein was available on the database. In the 46-1005 bp region, ecale is considered to be the isoform of human galectin-9.
It was 83% identical to ctin (GenBank accession number: AB005894) at the base level. Also, galectin-9 (GenB
ank accession number: NM # 002308.2).

In the homology search based on the amino acid sequence, the most homologous molecule is also the Sus scrofa Sus sc
rofa urate transporter / channel protein, 1
３313 amino acids showed high homology. In humans, ecalectin had 79.2% homology with amino acids 1 to 323 in the amino acid sequence, and was the highest homology in the galectin family (FIG. 3). Also, K
U-BL-50 showed high homology with many galectins. Characteristic amino acid sequences obtained by motif search include galactosidase binding signiture (galeptin sign
iture), suggesting that galectosidase binds. Galectin family ecalectin with homology, gale
ctin-4 (GenBank accession number: AB006781), gal
The homology with ectin-8 (GenBank accession number: X91790) is shown in FIG. Galectin-4 and galectin-8 are two ca
Two domains with rbohydrate-binding region (g
alectin-4-N terminal domain (Nt), galectin-4-C term
inal domain (Ct) and galectin-8-N terminal domain (N
t) and galectin-8-C terminal domain (Ct)), and it is known that they are linked by a link peptide (Yaron RH., JBC 270, 3447-53, 1994). From the results shown in FIG. 4, it was considered that KU-BL-50 and ecalectin also had two domains. (+) In the figure
Represents an amino acid that is commonly conserved in all galectins. Among them, (H-NPRF). (-V-
The amino acid combination of N). (WG) is commonly conserved in the galectin family, and this site is presumed to be a site to which galectin ligand binds.

From the above, KU-BL-50 also has an N-terminal domain and a C-terminal domain, and
It is considered that the nk peptide is connected.
Since the sequence common to galectins is clearly conserved in KU-BL-50, KU-BL-50 also appears to be a member of the galectin family. The galectin family has recently been considered to be associated with cancer, and KU-B
Since antibodies are present in various cancer patient sera such as L-50 cancer, such as an expression pattern restricted to cancer and bladder cancer, esophageal cancer, and renal cancer, there is a high possibility of expression in association with cancer. There are many cancer-related papers on galectin-1 and galectin-3, and it is thought that high expression of galectin-1 and decreased expression of galectin-3 are related to metastatic potential. Further Ho
It has been confirmed that galectin-9 is highly expressed in tumor cells in dgkin's disease. In colorectal cancer, galectin-4 may be a differentiation antigen. In prostate cancer, PTCA- expressed in prostate cancer but not expressed in normal prostate or benign prostatic hyperplasia than LNCap
No. 1 has been identified and is reported to be a member of the galectin family because of its sequence homology. These facts suggest that KU-BL-50 may be a member of the galectin family.

[0073]

According to the present invention, it is possible to provide a human bladder cancer antigen which can be applied to diagnosis and treatment of various cancers and tumors including bladder cancer, a gene encoding the same, and the like.

[0074]

[Sequence List] SEQUENCE LISTING <110> KEIO UNIVERSITY <120> Human Bladder Cancer Antigens <130> 000000101 <140> <141> <160> 27 <170> PatentIn Ver. 2.1 <210> 1 <211> 1566 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (46) .. (1035) <400> 1 ctagaatcca gcggccgctg aattctagaa ttcagcggcc gctga att cta ggc ggt 57 Ile Leu Gly Gly 1 gga gcg atg gcc ttc gg gcc cag gct ccc tac atg aac ccg atg 105 Gly Ala Met Ala Phe Arg Gly Ala Gln Ala Pro Tyr Met Asn Pro Met 5 10 15 20 gtc ccc ttc aca ggg atg atc caa ggg ggc ctc cag gac gga ctt cag 153 Val Pro Phe Thr Gly Met Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln 25 30 35 atc acc atc aat ggg acc gtt ctc atg tcc agt gga agc agg ttt act 201 Ile Thr Ile Asn Gly Thr Val Leu Met Ser Ser Gly Ser Arg Phe Thr 40 45 50 gtg aac ttg cag acc ggc cac agg gac aat gac att gcc ttc cac ttc 249 Val Asn Leu Gln Thr Gly His Arg Asp Asn Asp Ile Ala Phe His Phe 55 60 65 aac cct cgg ttc gaa gag ggc ggc tc gtg gtg aac acg aag cag 297 Asn Pro Arg Phe Glu Glu Gly Gly Tyr Val Val Cys Asn Thr Lys Gln 70 75 80 aat gga atc tgg ggg cct gag gag agg aag atg caa atg ccc ttc cag 345 Asn Gly Ile Trp Gly Pro Glu Glu Arg Lys Met Gln Met Pro Phe Gln 85 90 95 100 cgc gga cat cca ttt gag ctc agc ttc ctg gtc cag agc tcg cag ttc 393 Arg Gly His Pro Phe Glu Leu Ser Phe Leu Val Gln Ser Ser Gln Phe 105 110 115 cag gtg aca gtg aac ggg agg ctc ttt gtg cag tac acac cc Gln Val Thr Val Asn Gly Arg Leu Phe Val Gln Tyr Thr His Arg Val 120 125 130 ccc ttc cac cgg gtg gac acc atc tcc gtg aca ggc atc gtc cag ctg 489 Pro Phe His Arg Val Asp Thr Ile Ser Val Thr Gly Ile Val Gln Leu 135 140 145 tcc tac atc agc ttc cag cct cca ggt cgc tgg ccg gcc aac tcg gct 537 Ser Tyr Ile Ser Phe Gln Pro Pro Gly Arg Trp Pro Ala Asn Ser Ala 150 155 160 ccc att act caa acc gtc atc cac act gtg cag agc acc cct gga cag 585 Pro Ile Thr Gln Thr Val Ile His Thr Val Gln Ser Thr Pro Gly Gln 165 170 175 180 atg ttc cct aat ccc atg atc cca cct atg gca tat cct aac cca gtc 633 Met Phe Pro Asn Pro Met Il e Pro Pro Met Ala Tyr Pro Asn Pro Val 185 190 195 ttt ccc atc cct ttc ttc gcc tcc atc ccg ggc ggc ctg tac ccc tcc 681 Phe Pro Ile Pro Phe Phe Ala Ser Ile Pro Gly Gly Leu Tyr Pro Ser 200 205 210 aag agc atc atg gtg tcg ggc acc atc ctg ccc agc gct cag agt ttc 729 Lys Ser Ile Met Val Ser Gly Thr Ile Leu Pro Ser Ala Gln Ser Phe 215 220 225 tac atc aac ttg cgc tcg ggg agt gac atc gcc ttcac ccc 777 Tyr Ile Asn Leu Arg Ser Gly Ser Asp Ile Ala Phe His Leu Asn Pro 230 235 240 cgc ttc aag gag aac gcg gtg gtc cgc aac acg cag atc ggc agc tcc 825 Arg Phe Lys Glu Asn Ala Val Val Arg Asn Thr Ile Gly Ser Ser 245 250 255 260 tgg ggg cct gag gag cga ggc ctg ccc cgg aag atg ccc ttc tcc aga 873 Trp Gly Pro Glu Glu Arg Gly Leu Pro Arg Lys Met Pro Phe Ser Arg 265 270 275 ggc cag agc ttc ttg gtg tgg atc ctc tgt gaa agc cac tgc ttc aag 921 Gly Gln Ser Phe Leu Val Trp Ile Leu Cys Glu Ser His Cys Phe Lys 280 285 290 gtg gcc gtg gac ggt cag cac cta ttt gaa tac tac cac cgc Act ag ag A sp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Lys 295 300 305 cac ctg ccc acc atc aac tcc ctg gag gtg ggg ggc gac atc cag ctg 1017 His Leu Pro Thrh Ile Asn Ser Leu Glu Val Gly Gly Asp Ile Gln Leu 310 315 320 acg cac gtg cag cca tag acgagccccg ccctgggggt gaggcctggg 1065 Thr His Val Gln Pro 325 330 ccgaaaggct gtctgggtct gctcgtcacc ccccttccca ggctgccagg acctggggct 1125 tggctggaga gaccgtgtcc ttgcctggcc ctgcttctgg ctgcagcctc ccctggagcg 1185 gggaggacag ctggctggga ccgccttcct ccacctacgg aggaggtgcg gtctgcgcag 1245 gccggagccc cgtgttccct gcccccggcc tctgcctggg tcctggcctt cccaagctcc 1305 cagcccaggc ctgcccacac ctgcctctcc acccccccac ccggccttcc agtcacctag 1365 tcccaagcca ccagctgtca ctccacgtgg gaggcagtat cccctcttct ttctccaagc 1425 ttgaacctct ccttgcaccc tgcaccaact cttccccgcc ccggatagct gcctgaggac 1485 accccaccga tgggagcttt ctcccctttc cagcgtcctt ccaataaaga aagaaaactg 1545 ctaaaaaaaa aaaaaaaaaa a 1566 <210> 2 <211> 329 <212> PRT <213> Homo sapiens <400> 2 Ile Leu Gly Gly Gly Ala Met Ala Phe Arg Gly Ala Gl n Ala Pro Tyr 1 5 10 15 Met Asn Pro Met Val Pro Phe Thr Gly Met Ile Gln Gly Gly Leu Gln 20 25 30 Asp Gly Leu Gln Ile Thr Ile Asn Gly Thr Val Leu Met Ser Ser Gly 35 40 45 Ser Arg Phe Thr Val Asn Leu Gln Thr Gly His Arg Asp Asn Asp Ile 50 55 60 Ala Phe His Phe Asn Pro Arg Phe Glu Glu Gly Gly Tyr Val Val Cys 65 70 75 80 Asn Thr Lys Gln Asn Gly Ile Trp Gly Pro Glu Glu Arg Lys Met Gln 85 90 95 Met Pro Phe Gln Arg Gly His Pro Phe Glu Leu Ser Phe Leu Val Gln 100 105 110 Ser Ser Gln Phe Gln Val Thr Val Asn Gly Arg Leu Phe Val Gln Tyr 115 120 125 Thr His Arg Val Pro Phe His Arg Val Asp Thr Ile Ser Val Thr Gly 130 135 140 Ile Val Gln Leu Ser Tyr Ile Ser Phe Gln Pro Pro Gly Arg Trp Pro 145 150 155 160 Ala Asn Ser Ala Pro Ile Thr Gln Thr Val Ile His Thr Val Gln Ser 165 170 175 Thr Pro Gly Gln Met Phe Pro Asn Pro Met Ile Pro Pro Met Ala Tyr 180 185 190 Pro Asn Pro Val Phe Pro Ile Pro Phe Phe Ala Ser Ile Pro Gly Gly 195 200 205 Leu Tyr Pro Ser Lys Ser Ile Met Val Ser Gly Thr Ile Leu Pro Ser 210 215 220 Ala Gln Ser Phe Tyr Ile Asn Leu Arg Ser Gly Ser Asp Ile Ala Phe 225 230 235 240 His Leu Asn Pro Arg Phe Lys Glu Asn Ala Val Val Arg Asn Thr Gln 245 250 255 Ile Gly Ser Ser Trp Gly Pro Glu Glu Arg Gly Leu Pro Arg Lys Met 260 265 270 Pro Phe Ser Arg Gly Gln Ser Phe Leu Val Trp Ile Leu Cys Glu Ser 275 280 285 His Cys Phe Lys Val Ala Val Asp Gly Gln His Leu Phe Glu Tyr Tyr 290 295 300 His Arg Leu Lys His Leu Pro Thr Ile Asn Ser Leu Glu Val Gly Gly 305 310 315 320 Asp Ile Gln Leu Thr His Val Gln Pro 325 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: T3 <400> 3 aattaaccct cactaaaggg 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: T7 <400> 4 gtaatacgac tcacataggg c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-50 Sense Primer <400> 5 ctcatgtcca gtggaagcag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <2 23> Description of Artificial Sequence: KU-BL-50 Antisense Primer <400> 6 accaccgcgt tctccttgaa 20 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Beta -actin Sense Primer <400> 7 atctggcacc acaccttcta caatgagctg cg 32 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Beta-actin Antisense Primer <400> 8 cgtcatactc ctgcttgctg atccacatct gc 32 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-49 Forward Primer <400> 9 gccaccaacc catggaagat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-49 Reverse Primer <400> 10 ggctggagta tcccgtattg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-51 Forward Primer <400> 11 atacagctgg ggaggtagct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: KU-BL-51 Reverse Primer <400> 12 ggagaagctt gagatgctcc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-52 Forward Primer <400> 13 cccgcgccga tgtgaattat t 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-52 Reverse Primer <400> 14 ccgcatctcc agggtcttta a 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-53 Forward Primer <400> 15 aaggagctgc tacagccaca 20 <210> 16 < 211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-53 Reverse Primer <400> 16 gcacgctgaa gccataacag 20 <210> 17 <211> 22 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-49 Forward Primer <400> 17 caaagtggaa gccaagttca tc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-49 Reverse P rimer <400> 18 ctgccagaga tcttgaatag cc 22 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-51 Forward Primer <400> 19 aaccagaagg ccaagtggat 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: KU-BL-51 Reverse Primer <400> 20 cagaactgta gagcaagcgc ag 22 <210> 21 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Beta-actin Forward Primer <400> 21 tcacccacac tgtgcccatc tacga 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Beta-actin Reverse Primer <400> 22 cagcggaacc gctcattgcc aatgg 25 <210> 23 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Marathon cDNA Adapter1 <400> 23 aatacgactc actatagggc tcgagcggcc gcccgggcag gt 42 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: AP1 <400> 24 ccat cctaat acgactcact actatagggc 30 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: GSP1 <400> 25 cctcccgttc actgtcacct ggaactgc 28 <210> 26 <211> 23 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: AP2 <400> 26 actcactata gggctcgagc ggc 23 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: GSP2 <400> 27 tgttgcagac cacgtagccg ccctct 26

[Brief description of the drawings]

FIG. 1 shows the human bladder cancer antigen KU-BL-50mR of the present invention.
FIG. 3 is a view showing the results of examining NA expression by Northern blot.

FIG. 2: D of human bladder cancer antigen KU-BL-50 of the present invention
It is a figure which shows an NA sequence and an amino acid sequence.

FIG. 3 shows human bladder cancer antigens KU-BL-50 and ec of the present invention.
It is a figure which shows the comparison result of the amino acid sequence with alectin.

FIG. 4 shows human bladder cancer antigens KU-BL-50 and ga of the present invention.
It is a figure which shows the comparison result of the amino acid sequence with lectin-4, galectin-8, and ecalectin.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 13/10 C07K 14/47 4C084 35/00 16/30 4C085 C07K 14/47 19/00 4H045 16/30 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 C12P 21/08 1/21 C12Q 1/02 5/10 1/68 A C12P 21/08 G01N 33/15 Z C12Q 1 / 02 33/50 Z 1/68 33/53 D G01N 33/15 33/566 33/50 33/574 A 33/53 Z 33/566 33/577 B 33/574 C12N 15/00 ZNAA A61K 37/02 33 / 577 C12N 5/00 A F-term (reference) 2G045 AA26 AA40 CA18 CB01 CB02 DA12 DA13 DA14 DA36 DA78 FB02 FB03 4B024 AA12 BA31 BA36 BA80 CA01 CA04 CA09 CA11 CA12 DA06 EA03 GA11 HA01 HA12 4B063 QA01 QA05 QAQ QAQ QQ79 QR32 QR35 QR48 QR51 QR55 QR62 QR75 QR7 7 QR80 QR90 QS16 QS24 QS25 QS33 QS34 QS36 4B064 AG27 AG31 CA10 CA20 CC24 DA14 4B065 AA19X AA26X AA49X AA50X AA53X AA60X AA72X AA88X AA90X AA93A13 CA03 A03 CA25 A23 CA25 A03 CA25 A03 CA25 A23 BB01 CC03 4H045 AA11 AA20 AA30 BA10 BA41 CA40 CA41 DA75 DA76 DA86 EA51 FA72 FA73 FA74 HA05

Claims (28)

[Claims] 1. A DNA encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; Active protein 2. A DNA comprising the base sequence shown in SEQ ID NO: 1 or a sequence complementary thereto and a part or all of these sequences. 3. A DNA that hybridizes with the DNA according to claim 2 under stringent conditions and encodes a protein having immunity-inducing activity. 4. A protein comprising the amino acid sequence shown in SEQ ID NO: 2. 5. A protein comprising an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and having immunity-inducing activity. 6. A peptide comprising a part of the protein according to claim 4 and having immunity-inducing activity. 7. A fusion protein or fusion peptide in which the protein according to claim 4 or 5 or the peptide according to claim 6 is linked to a marker protein and / or a peptide tag. 8. An antibody which specifically binds to the protein according to claim 4 or 5 or the peptide according to claim 6. 9. The antibody according to claim 8, wherein the antibody is a monoclonal antibody. 10. A recombinant protein or peptide to which the antibody according to claim 8 or 9 specifically binds. A host cell comprising an expression system capable of expressing the protein or peptide according to any one of claims 4 to 6 or claim 10. 12. A non-human animal, wherein the function of a gene encoding the protein or peptide according to any one of claims 4 to 6 or 10 is deficient on a chromosome. 13. A non-human animal, which overexpresses the protein or peptide according to any one of claims 4 to 6 or claim 10. 14. The non-human animal according to claim 12, wherein the non-human animal is a mouse or a rat. 15. Using a test substance, any one of claims 4 to 6, or the protein or peptide according to claim 10 and a T cell, measuring and evaluating immunity-inducing activity in the T cell. A method for screening for a substance that promotes or suppresses immunity inducing activity. 16. Use of a test substance, the host cell according to claim 11, and a T cell to measure / evaluate the immunity-inducing activity of said T cell. Screening method. 17. The method according to claim 11, wherein the host cell according to claim 11, which is transfected with both a vector expressing the HLA and a vector expressing the test polypeptide, and the T cell.
A method for screening for a substance that promotes or suppresses immunity inducing activity, comprising measuring and evaluating immunity inducing activity in cells. 18. The method according to claim 1, which is capable of expressing HLA transfected with a vector expressing the test polypeptide.
A method for screening for a substance that promotes or suppresses immunity inducing activity, comprising using the host cell according to 1 and T cells to measure and evaluate immunity inducing activity in said T cells. 19. An immune-inducing activity promoting method comprising administering a test substance to the non-human animal according to any one of claims 12 to 14, and measuring and evaluating the immunity-inducing activity on T cells of said non-human animal. Or a method of screening for an inhibitor. 20. The screening for a substance that promotes or suppresses the immunity-inducing activity according to any one of claims 15 to 19, wherein the measuring and evaluating the immunity-inducing activity is measuring and evaluating the interferon-γ activity in T cells. Method. 21. An immunity-inducing activity-promoting substance obtained by the screening method according to any one of claims 15 to 20. A immunity-inducing activity-suppressing substance obtained by the screening method according to any one of claims 15 to 20. 23. The protein or peptide according to any one of claims 4 to 6, or claim 10, or claim 2.
An antitumor agent comprising the substance for promoting immunity-inducing activity according to 1 as an active ingredient. 24. The protein or peptide according to any one of claims 4 to 6, or claim 10, or claim 2.
An activated T cell induced by in vitro stimulation with the immunity-inducing activity promoting substance according to 1. 25. D encoding the protein or peptide according to any one of claims 4 to 6 or claim 10.
Bladder cancer, brain tumor, malignant melanoma, chronic myelogenous leukemia, acute myelogenous leukemia, lymphoma, esophagus cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, breast cancer, consisting of all or part of the antisense strand of NA or RNA, Colorectal cancer, stomach cancer, liver cancer, gallbladder cancer,
Probe for diagnosis of testicular cancer, uterine cancer, ovarian cancer, sarcoma. 26. A bladder cancer, a brain tumor, a malignant melanoma, a chronic myelogenous leukemia, an acute bone marrow comprising the diagnostic probe for cancer according to claim 25 and / or the antibody according to claim 8 or 9. Diagnostic agent for leukemia, lymphoma, esophagus cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, breast cancer, colon cancer, stomach cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, sarcoma. 27. A bladder cancer cell cDNA library, λ phage cD, prepared by preparing a cDNA using mRNA obtained from a bladder cancer cell line, introducing the cDNA into a λ phage vector and infecting Escherichia coli.
An antitumor agent, comprising a bladder cancer cell-derived bladder cancer antigen, obtained by screening an NA clone using a bladder cancer patient serum. 28. A bladder cancer cell cDNA library, λ phage cD, prepared by preparing a cDNA using mRNA obtained from a bladder cancer cell line, introducing the cDNA into a λ phage vector and infecting Escherichia coli.
All or part of the antisense strand of DNA or RNA encoding a bladder cancer antigen derived from a bladder cancer cell, or a bladder derived from the bladder cancer cell, obtained by screening an NA clone using a bladder cancer patient serum A diagnostic agent for detecting a tumor, comprising an antibody against a cancer antigen.