WO1998022582A1

WO1998022582A1 - Human cancer regression antigen protein

Info

Publication number: WO1998022582A1
Application number: PCT/JP1997/004182
Authority: WO
Inventors: Kyogo Itoh; Yasuhisa Imai; Uhi Tho
Original assignee: Kyogo Itoh; Yasuhisa Imai; Uhi Tho
Priority date: 1996-11-21
Filing date: 1997-11-17
Publication date: 1998-05-28
Also published as: AU4966797A

Abstract

A tumor antigen protein, a tumor antigen peptide corresponding thereto, or the like, which can be applied to a wide variety of tumors including squamous cell carcinoma and, even when treatable tumors are limited, can be applied to a major part of patients among those suffering from such tumors, or can be applied to various tumors while supplementing this method with tumor immunization or vice versa. They can activate antitumor immunization, treat autoimmune diseases, and provide a diagnostic method for tumors and autoimmune diseases.

Description

Description Human cancer regression antigen protein

Technical field

The present invention belongs to the medical field, and more specifically, a method for treating cancer or an autoimmune disease, and more specifically, a cancer regression antigen that is regressed by being attacked by cytotoxic T cells, and an immunotherapy using the same. About.

It is known that the immune system, especially T cells, plays an important role in eliminating tumors by living organisms. Indeed, infiltration of lymphocytes, which show cytotoxic activity against tumor cells, was observed in human tumors (Arch. Surg. 126: 200-205, 1990), and cells that recognize their own tumor cells from melanoma Toxic T cells (CTL) have been isolated (Immunol. Today 8: 385, 1987, J. Immunol. 138: 989, 1987. Int. J. Cancer 52: 52-59, 1992, etc.). The clinical results of melanoma treatment with T cell transfer also suggest the importance of T cells for tumor elimination (J. Natl. Cancer. Inst. 86: 1159, 1994).

CTLs that attack their own tumor cells recognize their complexes of tumor antigen peptides with major histocompatibility complex (MHC) class I antigens using the T cell receptor (TCR), Is attacking. This tumor antigen peptide is produced by the synthesis of tumor antigens (proteins) in cells and the subsequent degradation by proteosomes into peptides in the cytoplasm. On the other hand, MHC class I antigen binds to the above-mentioned tumor antigen peptide, moves to transgolgi on the mature side via cis Golgi, and is expressed on the cell surface (clinical immunity 27 (9): 1034-1042, 1995). Background art

A tumor antigen protein, presented on MHC class I antigen on human cancer cells and targeted by host T cells, was identified by T. Boon in 1991 (Science 254: 1643-164 7,1991). This antigen causes cancer cells that express this antigen to be attacked by CTLs. It is called a cancer regression antigen because it regresses, and is named Melanoma antigen (MAGE) because it was identified from melanoma cells.

Subsequently, tumor antigen proteins recognized by CTL were successively identified from melanoma cells and the like. Tumor antigen proteins identified so far are classified into one of the following four categories according to their origin, structure (presence or absence of mutation) and expression mode (T. Bon et al., J. Exp. Med. 183). : 725-729, 1996):

i) Tumor-Specific Shared Antigens

The antigens classified here are expressed only in the testis and placenta in normal tissues, and are widely expressed in tumor tissues such as melanoma, head and neck cancer, non-small cell lung cancer, and bladder cancer. It is protein. Examples of this category of tumor antigen proteins include the above-mentioned MAGE and its one or more proteins that form two or more similar families (J. Exp. Med. 178: 489-495, 1993). ), BAGE (Immunity 2: 167-175, 1995) and GAGE (J. Exp. Med. 182: 689-698.1995), all of which have been identified from melanocytic cells. Recently, a widespread expression of NA17-A in melanoma was reported. It is translated from the intron portion of the N-acetylglucosaminyltransferase V gene, and the HLA-A2-restricted antigen peptide (VLPOVF IRC) is expressed as a cancer regression antigen and recognized by CTL. You. ii) Differentiation antigens

Tumor antigen proteins classified here are expressed in melanocytes in normal tissues and are a group of proteins expressed only in melanomas in tumor tissues. These tissue-specific proteins are strongly expressed in tumor cell melanomas. They are not found in other tissue types of cancer (adenocarcinoma or squamous cell carcinoma). It is considered a tumor antigen protein. Tumor antigen proteins in this category include tyrosinase (J. Exp. Med. 178: 489-495, 1993), MART-1 (Proc. Natl. Acad. Sci. USA 91: 3515, 1994), gp 1 0 0 (J. Exp. Med. 179: 1 005-1009, 1994) and gρ75 (J. Exp. Ed. 181: 799-352, 1995), and these genes are all cloned from melanoma cells. In addition, Melan-A (J. Exp. Med. 180: 35, 1994) was identified, but it was later found to be the same molecule as MART-1.

In addition, since the antigens classified here are also expressed in normal melanosite, there is a possibility that they may be used as target molecules in melanosite-destructive autoimmune diseases. In particular, MART-1 Zmelan-A is considered to be a target molecule in von-Koyanagi-Ichiharada disease (S. Sugita, et al., Int. Immunol. 8: 799-803, 1996). gp100 may act as an in vivo cancer regression antigen because the melanoma expressing it is highly sensitive to immunotherapy.

This category 1 tumor antigen protein is not expressed in tumors other than melanomas and cannot be applied to other tumors.

i i i) Antigens Specific for Individual Tumors

Tumor antigen proteins classified into this region are cancer-specific new antigens associated with genetic changes that occur during the transformation of normal cells into cancer. Gene changes are known to be point mutation (mutant CDK4 antigen, mutation; 8-catenin antigen, MUM antigen) and alterative open reading frame (mutant gp75 antigen). Therefore, it is considered that such an antigen is specific to cancer and that specific immunity is easily established. On the other hand, most of the genetic changes are expressed only in individual tumors or individual tumor cells. Therefore, it has the drawback that its expression frequency is extremely low and it is difficult to apply it clinically as a vaccine molecule for cancer treatment.

iv) Ubiqui tous Antigens

P15 is known as a case in which an antigen that is universally expressed as a non-mutant (ubiqui tous) in most normal cells and cancer cells is a cancer recognition molecule of CTL. The p15 molecule has an HLA-A24 binding peptide. pi5 is more strongly expressed in cancer cells than in normal cells. From that point of view, the HER-2Zneu antigen, which is an oncogene protein that is strongly expressed in adenocarcinoma and the like, is also classified as the same. That is, HER-2 / neu has an HLA-A2-binding peptide and is recognized by host killer T cells as a cancer regression antigen.

If the antigen classified here is tumor antigen protein, it is considered to be applicable to a wide range of cancers due to its ubiquitous expression, but it will damage normal tissues due to poor disease specificity There is a possibility that CTL induction may be difficult (because of trans).

It was reported that MUC-11 antigen-specific CTL, which was thought to be MHC-unrestricted, recognized the peptide derived from the antigen, STAPPAHGV, as HLA-A11-restricted, and clinical studies using MAGE-3 peptide Initial results of the trial were reported (M. Marchand et al., Int. J. Cancer 63: 883-885, 1995). These suggest the possibility of developing a cancer vaccine using a cancer regression antigen.

Most of the above identified antigen peptides except HER-2 / neu have been found in melanomas, and have not been reported in squamous cell carcinomas or adenocarcinomas with high incidence. However, we recently reported that HL A2661-restricted cancer antigen, a peptide, is present and recognized by host CTLs from esophageal squamous cell carcinoma (M. Nakao, et a Cancer Res. 55: 4248-4252, 1995). Therefore, it is suggested that squamous cell carcinoma also has a tumor antigen protein that encodes a similar antigen peptide. Squamous cell carcinoma is one of the most common human cancers.It is known that squamous cell carcinoma in esophageal and lung cancers is relatively resistant to current chemotherapy and radiation therapy. I have. From this point of view, the development of specific immunotherapy using tumor antigen peptides is expected. Disclosure of the invention The object of the present invention is to be applicable to a wide range of tumors such as squamous cell carcinoma, or to a large number of patients with tumors, even if the applicable tumors are limited, or to treat or diagnose such tumors. An object of the present invention is to provide a tumor antigen peptide and the like which assist complementation of cancer and can be applied to various tumors.

On the other hand, tumor antigen proteins that are highly expressed in tumors, on the other hand, are also thought to be expressed in normal tissues and cause an autoimmune disease due to excessive immune responses derived from the tumor antigen proteins. I have. For example, it has been reported that when melanomas are treated with a combination of a chemotherapeutic agent and IL-2, vitiligo symptoms appear (J. Clin. Oncol. 10: 1338-1343, 1992). ). This is because CTLs or antibodies are induced and produced against the tumor antigen protein fragment peptide and MHC complex appearing in melanoma, and they act on normal skin tissue, causing symptoms similar to autoimmune disease. This is probably due to the appearance of vitiligo symptoms.

When an autoimmune disease develops due to excessive induction of specific immunity derived from a tumor antigen protein, the antisense DNA or tumor antigen peptide that prevents expression of a gene that encodes the tumor antigen protein is blocked. A therapeutic method that specifically blocks the immune response using evening gonists is expected.

Identification of tumor antigen proteins from squamous cell carcinomas to obtain tumor antigen proteins or their corresponding tumor antigen peptides that can be widely applied to the treatment and diagnosis of tumor cells other than melanoma cells, especially squamous cell carcinoma, etc. Tried.

The present inventors have established CTL (YAM-CTL) that recognizes an HLA-CW0102-restricted tumor antigen peptide from metastatic lymph node cancer tissue infiltrating lymphocytes of esophageal cancer patients. This CTL strongly impairs the HLA-CW0102-positive esophageal cancer cell line TE8 (Kumamoto University School of Medicine).

Therefore, the recombinant plasmid of the cDNA library and the recombinant plasmid of the HLA-CW0102 cDNA created from the TE8 cancer cell line were simultaneously transfected into the fibroblast cell line C0S7 cells, and YAM-CTL was added to the transfectant. The cells were allowed to act, and whether or not the YAM-CTL cells were activated was screened by measuring the amount of IFN-7 produced. The result As a result, a gene encoding the tumor antigen protein of the present invention was successfully cloned from tumor cells other than melanoma TE8.

That is, in one embodiment, the present invention relates to a partial peptide of a tumor antigen protein having an amino acid sequence of SEQ ID NO: 2, which binds to a major histocompatibility complex (MH C) class I antigen and has T cells. A tumor antigen peptide or a derivative thereof, a drug containing the tumor antigen peptide or a derivative thereof according to the present invention; an antibody against the tumor antigen peptide or a derivative thereof of the present invention; a tumor antigen peptide or the present invention An oligonucleotide molecule encoding the derivative thereof, preferably the oligonucleotide molecule whose nucleotide sequence encoding a tumor antigen protein is the nucleotide sequence of SEQ ID NO: 2; the tumor antigen protein of SEQ ID NO: 2; Consists of the coding sequence of the coding polynucleotide molecule or a sequence complementary to the base sequence in its 5 'non-coding sequence An oligonucleotide molecule or a chemically modified product thereof; a medicament containing the oligonucleotide molecule of the present invention; a plasmid having the oligonucleotide molecule of the present invention; and a transformant transformed with the plasmid of the present invention. .

In another embodiment, the present invention relates to a partial peptide of a tumor antigen protein having the amino acid sequence of SEQ ID NO: 4, which is recognized by T cells by binding to a major histocompatibility complex (MHC) class I antigen. A tumor antigen peptide or a derivative thereof to be used; a medicament containing the tumor antigen peptide or a derivative thereof of the present invention; an antibody against the tumor antigen peptide of the present invention or a derivative thereof; and a tumor antigen peptide of the present invention or a derivative thereof. An oligonucleotide molecule that encodes, preferably the oligonucleotide nucleotide sequence whose nucleotide sequence encodes a tumor antigen protein; SEQ ID NO: 4; An oligonucleotide consisting of a sequence complementary to the base sequence in the coding sequence of the coding polynucleotide molecule or its 5 'non-coding sequence. Reochido molecule or chemical modifications thereof; pharmaceutical containing Origonu Kureochido molecules of the present invention; bra with O oligo nucleotide molecule of the present invention And a transformant transformed with the plasmid of the present invention. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequences of fragments 1 and 2 obtained from clone 3. Underline indicates fragment 1 and bold diagonal line indicates fragment 2.

FIG. 2 is a graph showing the amount of IFN- ₇ produced by fragments 1 and 2 obtained from clone 3 measured by EUSA. ORF indicates open reading frame, F 1 indicates fragment 1, and F 2 indicates fragment 2. (-) Indicates that the cotransfected plasmid was not transfected, A24 indicates the cotransfected plasmid of HLA-A24 cDNA, and Cwl indicates the recombinant plasmid of HLA-Cw0102 cDNA. The result of simultaneous transfection is shown.

FIG. 3 shows the amino acid sequences of fragments 1, 2 and 3 obtained from clone 6. Underline indicates fragment 1, bold italic indicates fragment 2, double line indicates fragment 3.

FIG. 4 is a graph showing the amount of IFN-7 produced by fragments 1, 2 and 3 obtained from clone 6 measured by ELIS A. ORF indicates the open reading frame, F1 indicates fragment 1, F2 indicates fragment 2, and F3 indicates fragment 3. (1) is not simultaneously transfected, A24 is a simultaneously transfected recombinant plasmid of HLA-A24 cDNA, and Cwl is a simultaneously transfected plasmid of HLA-Cw0102 cDNA. Shows what was transfected. BEST MODE FOR CARRYING OUT THE INVENTION

The meaning of the terms used in this specification will be clarified, and embodiments of the invention will be described.

"Partial peptide of tumor antigen protein, tumor antigen peptide recognized by T cells by binding to major histocompatibility complex (MHC) class I antigen" A partial peptide consisting of at least 7-10, preferably 9 contiguous amino acid sequences of a tumor antigen protein, which is presented on the cell surface in association with the MHC class I antigen on the cell surface In this case, when a T cell that specifically binds to the conjugate binds, it can transmit a signal to the T cell, that is, form a conjugate with the MHC class I antigen recognized by the T cell. Means peptide. The “bond” here is a non-covalent bond.

Methods for confirming that a peptide binds to MHC class I antigen and is recognized by T cells include, for example, endogenous expression of the peptide in appropriate cells, or by externally adding (pulsing) the peptide. The peptide is presented on the cell surface by binding to the MHC class I antigen, followed by the action of tumor antigen protein-specific T cells on the peptide-presenting cells, which damage the peptide-presenting cells There is a method to measure the site force in produced when receiving the drug (interferon, TNF, and CTL). Further, as a method for measuring the cytotoxicity of peptide presentation cells, it can also be used how to use a peptide-presenting cells labeled with ^{5 1} C r. Here, CTLs are preferably used as T cells to be recognized.

The tumor antigen protein or tumor antigen peptide according to the present invention can be identified, for example, as follows.

To identify them, first prepare a set of tumor cells with matching MHC-class I alleles and CTLs that attack these cells.

Next, the tumor antigen peptide that binds to the MHC class I antigen of the tumor cells is oxidized and extracted, and various peptides separated by high-performance liquid chromatography express antigen-presenting MHC. By pulsing cells that do not express the tumor antigen protein (for example, B cells of the same patient), and examining the CTL response, the tumor antigen peptide was identified, and then mass spectrometry was performed. This method is used to determine the sequence. By this method, the same amount of gp100 from melanoma cells A tumor antigen peptide derived from offspring Pmell7 has been identified (Science264: 716-719, 1994).

) 0

Alternatively, unlike the method for directly identifying a tumor antigen peptide as described above, there is also a method for determining a gene encoding a tumor antigen protein and further identifying the corresponding tumor antigen peptide. This involves cloning the gene encoding the tumor antigen protein using molecular biology techniques. CDNA is prepared from tumor cells, and the cDNA is transfected with cells that do not express the tumor antigen protein (for example, COS cells) together with the antigen-presenting MHC class I antigen gene to express them temporarily. Screening based on the reactivity of the CTL against it is repeated to isolate the gene encoding the tumor antigen protein. By this method, the above-mentioned genes of MAGE, tyrosinase, MART-1, gp100, and gp75 have been cloned.

The following method is used to estimate and identify the tumor antigen peptide actually bound to and presented to the MHC class I antigen from the information on the tumor antigen gene. First, fragments of genes encoding tumor antigen proteins of various sizes were prepared by PCR, exonuclease, restriction enzymes, etc., and cells that did not express tumor antigen proteins together with antigen-presenting MHC class I antigen genes ( For example, transfected into COS cells, etc., and transiently expressed, and the region containing the tumor antigen peptide is limited by the reactivity of CTL. After that, the peptide was synthesized, and the antigen-presenting MHC class I antigen was expressed, but it was pulsed into cells that did not express the tumor antigen protein, and the tumor antigen peptide was identified by examining the reaction of cτL. Med. 176: 1453, 1992, J. Exp. Med. 179: 24, 759, 1994).

For the types of MHC class I antigens such as HLA-Al, -A0201, -A0205, -All, A31, -A6801, -B7, -B8, -B2705, -37, -CW0401, and -CW0602, binding The sequence regularities (motifs) of the peptides presented are known (seminars in IMMUNOLOGY 5: 81-94, 1993), and the tumor antigen peptide candidates are examined with reference to them. A method in which the peptide is bound and confirmed by the same method as described above is also used (Eur. J. Immunol, 24: 759, 1994, J. Exp. Med. 180: 347, 1994).

The peptides determined in this way can be produced by methods known in normal peptide chemistry. For example, "Peptide Syntheses", Interscience, New York. 1996, "The Proteins", Vol. 2, Academic Press Inc., New York. 1976. "Peptide synthesis" Maruzen Co., Ltd., 1975, "Peptide Synthesis Basics and Experiments ", Maruzen, 1985, and the like. That is, either the liquid phase method or the solid phase method can be selected for synthesis depending on the configuration of the C-terminal site, and the liquid phase method is more preferable. That is, a peptide can be produced by appropriately protecting and deprotecting a functional group of an amino acid with an appropriate protecting group, and bonding the amino acid to each residue or several residues. The protective group for the functional group of the amino acid is described, for example, in the above-mentioned book describing peptide chemistry.

In the present specification, “a derivative of the tumor antigen peptide of the present invention (a derivative of a tumor antigen peptide that is a partial peptide of a tumor antigen protein and that is recognized by T cells by binding to an MHC class I antigen)” Means a peptide in which one or several amino acid sequences of the tumor antigen peptide of the present invention have been substituted, deleted, inserted or added. Preferred derivatives include the tumor antigen peptides, in which the epitope region involved in binding to CTL remains unchanged and amino acid residues involved in binding to MHC class I antigen are substituted, deleted, or deleted. Inserted or added derivatives can be mentioned, and more preferably, derivatives thereof, in which only one amino acid residue is substituted (Im munol. 84: 298-303, 1995). Such a derivative can bind strongly to the MHC class I antigen while maintaining the binding property to CTL as it is, so that it can be applied as a more useful tumor antigen peptide.

Such a derivative can be prepared, for example, by the method described in Molecular Cloning: A Laboratory Manual, 2nd edition, Volume 13, Sambrook, J. et al., Cold Spring Harber Labolatory Press, New York, 1989. Site-directed mutagenesis And can be prepared by a method such as PCR.

Therefore, the tumor antigen peptide of the present invention or a derivative thereof is encoded by the below-described oligonucleotide of the present invention.

In addition, the “derivative of the present invention” includes a derivative of the tumor antigen peptide of the present invention or a derivative in which a partial amino acid residue of the peptide is substituted, deleted, inserted, or added, wherein the amino group or the carboxyl group is modified. Are also included.

Examples of the modifying group for the amino group include an acyl group.Specific examples include an alkanol group having 1 to 6 carbon atoms, 1 carbon atom substituted by a phenyl group, an alkanol group having 6 or more carbon atoms, and 5 to 5 carbon atoms. A carbonyl group substituted with 7 cycloalkyl groups, an alkylsulfonyl group having 1 to 6 carbon atoms, a phenylsulfonyl group, and the like.

Examples of the carboxyl group-modifying group include an ester group and an amide group. Specific examples of the ester group include an alkyl ester group having 1 to 6 carbon atoms, a carbon atom having 0 carbon atoms substituted with a phenyl group, Examples of the amide group include an amide group, one or two alkyl groups having 1 to 6 carbon atoms, and a cycloalkyl ester group having 5 to 7 carbon atoms. Substituted amide group, phenyl group-substituted alkyl group having 0 to 6 carbon atoms, amide group substituted with 1 or 2 carbon atoms, 5- to 7-membered ring including amide group nitrogen atom Amide groups forming azacycloalkanes and the like can be mentioned.

The present invention further provides a medicament containing the tumor antigen peptide of the present invention or a derivative thereof. The tumor antigen protein and the tumor antigen peptide of the present invention can be administered together with an adjuvant so as to effectively establish cellular immunity, or can be administered in the form of particles. As the adjuvant, those described in the literature (Cl in. Microbiol. Rev. 7: 277-289. 1994) can be applied. In addition, dosage forms include ribosome preparations, particulate preparations bound to beads with a diameter of several meters, and preparations linked to lipids to efficiently present antigen to MHC class I antigens. Any method of administration is used. In addition, dendritic pongee pulsed with tumor antigen peptide It is also conceivable to administer antigen-presenting cells such as vesicle microphages or cells transfected with DNA encoding tumor antigen protein. The dosage of the tumor antigen protein and tumor antigen peptide of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, etc., and is usually 0.0001 nig to 1000 nig, preferably 0. .000 mg to 1000 mg, preferably administered once every several days to several months.

The “antibody” against the tumor antigen peptide or a derivative thereof according to the present invention may be, for example, a tumor antigen by a method described in Antibodies; A Laboratory Manual, Lane. HD et al., Edited by Cold Spring Harber Laboratory Press, New York 1989. By immunizing an appropriate animal with an appropriate method using the protein or its fragment peptide, an antibody that recognizes a tumor antigen protein or an antibody that neutralizes its activity can be easily prepared. Uses of the antibody include affinity chromatography, screening of a cDNA library, immunological diagnostic methods, pharmaceuticals and the like. The immunological diagnostic method can be appropriately selected from an immunoblot method, a radioimmunoassay method (RIA), an enzyme immunoassay method (ELISA), a fluorescence or luminescence measurement method, or the like.

The present invention further relates to an oligonucleotide molecule encoding the tumor antigen peptide of the present invention or a derivative thereof. The oligonucleotide molecules of the present invention can be in the form of DNA or RNA, where DNA includes cDNA, genomic DNA and synthetic DNA. Also, DNA and RNA may be single-stranded or double-stranded, and in the case of single-stranded, may include both the sense strand and the antisense strand.

Oligonucleotide molecules in which a part of a certain nucleotide sequence has been substituted, deleted, inserted or added are described in Molecular Cloning: A Laboratory Manual, 2nd edition, Volume 1-3, Sambrook, J. et al., Cold Spring Harber Labolatory It can be produced by the method described in Press Publishing New York, 1980. For example, it can be produced by site-directed mutagenesis or PCR. The oligonucleotide molecules of the present invention also include these mutant oligonucleotide molecules. Examples of such mutant oligonucleotide nucleotides include, for example, one or more nucleotides in the nucleotide sequence of SEQ ID NO: 2 or 4. Oligonucleotide molecules in which three bases have been substituted, deleted, inserted or added. The oligonucleotide molecules of the present invention also include "oligonucleotide molecules that hybridize to the oligonucleotide molecules of the present invention under stringent conditions". Taking a DNA molecule as a typical example of the oligonucleotide molecule, “a DNA molecule that hybridizes to a DNA molecule” can be obtained, for example, by the method described in the aforementioned Molecular Cloning. Here, “hybridizing under stringent conditions” means, for example, heating at 42 ° C. in a solution of 6 XSSC, 0.5% SDS and 50% formamide, followed by 0.1 XSSC Under the condition of washing at 68 ° C. in a 0.5% SDS solution, a positive hybridization signal was still observed.

In addition, by expressing the DNA encoding the tumor antigen peptide of the present invention, it becomes possible to produce the tumor antigen peptide in large quantities.

Expression of DNA to produce a protein can be carried out, for example, based on many books and literature such as the aforementioned Molecular Cloning. A translation initiation codon is added upstream of the DNA to be expressed and a translation stop codon is added downstream to control the promoter sequence (eg, trp, lac, T7, SV40 early promoter) that controls transcription. By adding the gene and incorporating it into an appropriate vector (for example, pBR322, pUC19, pSV • SPORT1, etc.), an expression plasmid that replicates and functions in host cells is produced. Next, the expression plasmid is introduced into an appropriate host cell to obtain a transformant cell. Examples of the host cell include prokaryotes such as Escherichia coli, unicellular eukaryotes such as yeast, and cells of multicellular eukaryotes such as insects and animals. Methods for introducing genes into host cells include the calcium phosphate method, the DEAE-dextran method, and the electric pulse method. The transformant produces the target protein by culturing in a suitable medium. The protein obtained as described above can be isolated and purified by a general biochemical method.

Transformants transformed with the plasmid of the present invention are also within the scope of the present invention. Is included.

The present invention further relates to a medicament containing the oligonucleotide molecule of the present invention. The “medicine” containing the oligonucleotide molecule of the present invention can treat or prevent a tumor by, for example, administering the DNA of the present invention to a tumor patient or the like. Methods of administering the DNA of the present invention and introducing it into cells include viral vectors and other methods (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1) 23-48 (1994). Any of the methods in Experimental Medical Special Edition, 12 (15), (1994), and references cited therein (etc.) can be applied.

As a method using a viral vector, for example, the DNA of the present invention is added to an RNA virus or DNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, polio virus, and simbis virus. There is a method of incorporating and introducing. Among them, a method using retrovirus, adenovirus, adeno-associated virus, vaccinia virus and the like is particularly preferable.

Other methods include direct injection of the expression plasmid into the muscle (DNA-injection method), ribosome method, lipofectin method, microinjection method, calcium phosphate method, and electoral poration method. The ribosome method is preferred.

Plasmids having these oligonucleotide nucleotide molecules of the present invention are also included in the scope of the present invention.

In order for the gene encoding the tumor antigen peptide of the present invention to actually act as a medicament, an in vivo method of directly introducing the gene into the body, and the use of certain cells from humans to transform the gene in vitro There is an ex vivo method to introduce cells into cells and return the cells to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1) 23-48 (1994), extra edition of experimental medicine, 12 (15), (1994), and references cited therein). In vivo methods are more preferred. When administered by the in vivo method, it can be administered by an appropriate administration route depending on the disease, symptom and the like for the purpose of treatment. For example, it can be administered intravenously, intraarterially, subcutaneously, or intramuscularly. When administered by the in vivo method, for example, it can be in the form of a liquid preparation or the like.However, it is generally an injection containing the DNA of the present invention as an active ingredient. A carrier may be added. In addition, the DNA-containing ribosome or membrane fusion ribosome (such as Sendai virus (HVJ) -liposome) of the present invention is in the form of a ribosome preparation such as a suspending agent, a freezing agent, and a centrifugal concentrated freeze agent. be able to.

The content of the DNA of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, etc., and usually, the DNA of the present invention is 0.0001 mg to 100 mg, preferably O.OOlmg. 1010 mg, preferably administered once every few days or months.

The present invention further provides an oligonucleotide molecule comprising a coding sequence of a polynucleotide molecule encoding the tumor antigen protein of SEQ ID NO: 2 or 4, or a sequence complementary to a base sequence in the 5 ′ non-coding sequence thereof, or It relates to the chemically decorated body. Preferably, a DNA consisting of 9 or more bases having a sequence complementary to the base sequence in the coding sequence of the polynucleotide molecule consisting of the base sequence of SEQ ID NO: 2 or 4 (structural gene portion) or its 5 ′ non-coding sequence Or RNA. Such DNA or RNA is the DNA of the antisense strand of double-stranded DNA or RNA corresponding to the DNA of the antisense strand, and is composed of 9 bases or more (hereinafter referred to as antisense oligonucleotide). Say.

This antisense oligonucleotide can be easily prepared, for example, as DNA based on the nucleotide sequence of the gene encoding the tumor antigen peptide of the present invention, or by incorporating this DNA into a gene expression plasmid in the antisense direction. Can be produced.

This antisense oligonucleotide encodes the cDNA of the gene of the present invention. It may be a sequence complementary to any of the inking portion and the 5 'non-coding portion, but is preferably a transcription initiation site, a translation initiation site, a 5' untranslated region, a boundary region between exon and intron, or 5 '. It is desirable that the sequence be complementary to the CAP region.

“Chemical modification of an oligonucleotide molecule” refers to a chemically modified product that can enhance the translocation of DNA or RNA into cells or the stability in cells. For example, phosphothioate, phosphorodithioate Derivatives such as alkyl phosphotriesters, alkyl phosphonates and alkyl phosphoamidates ("Ant isense

RNA and DNA "WILEY — USS, 1992, P. 1-50). This chemically-modified product can be produced according to the same literature.

The expression of the gene encoding the tumor antigen protein can be controlled using DNA or RNA (hereinafter, referred to as antisense oligonucleotide) having a complementary sequence of the present invention. This method can treat or prevent autoimmune diseases by reducing the production of tumor antigen proteins. Pharmaceuticals containing such antisense oligonucleotides are also included in the present invention.

When the antisense oligonucleotide is administered as it is, a preferable length of the antisense oligonucleotide is, for example, 5 to 200 bases.

When an antisense oligonucleotide is incorporated into an expression plasmid, preferred lengths of the antisense oligonucleotide include, for example, 100 bases or more, and preferably 300 bases or more, More preferably, it is 500 bases or more.

When incorporating an antisense oligonucleotide into an expression plasmid, examples of a method for introducing this antisense oligonucleotide into cells include the method described in Experimental Medicine, Vol. 12, 1994, and Liposome Co., Ltd. There is a method using a replacement virus. Expression plasmid for antisense oligonucleotides The gene is simply connected by connecting the gene of the present invention so that the gene of the present invention is transcribed in the reverse direction behind the promoter using a normal expression vector, that is, in the 3 ′ to 5 ′ direction. Can be manufactured.

A plasmid having such an antisense oligonucleotide is also included in the present invention.

When the antisense oligonucleotide or its chemically modified product is administered as it is, it is formulated by mixing with a stabilizer, buffer, solvent, etc., and is used simultaneously with antibiotics, anti-inflammatory drugs, anesthetics, etc. You can also. The preparations thus prepared can be administered in various ways. Preferably, the administration is performed daily or every few days to several weeks. In order to avoid such frequent administration, it is possible to prepare a sustained-release minipellet preparation and implant it near the affected area. Alternatively, it can be continuously and gradually administered to a patient using an osmotic pump or the like. The usual dosage should be adjusted so that the concentration at the site of action is 0.1%.

A medicament containing such an antisense oligonucleotide or a chemically modified product thereof is also included in the present invention. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Reference example 1

Establishment of cytotoxic T cell (CTL) strain against esophageal cancer cell line

The operatively removed 67-year-old male esophageal cancer metastatic lymph node tissue was aseptically minced and pasted into a paste, and then in the presence of interleukin 2 for about 60 days, 5% CO 2 (95) Incubate in an incubator. Meanwhile, culture 2 8 after day cytotoxic ability against various cancer cells of T cells to proliferate in culture frequently ^{5 1} C r release assay and IFN - were analyzed by 7 measurements. As a result, T cells from day 39 to day 49 of culture were mainly CD8-positive killer T cells, and were restricted by HLA-Cw0102 among MHC class I antigens. Was found to exhibit CTL activity. Among the HLA-Cw0102-positive cancer cells, the squamous cell carcinoma TE8 cell line (M. Nakao et al., Cancer Research 55, 4248-4252, 1995) showed the highest sensitivity to the CTL. Therefore, a large amount of the above CTL (named YA-CTL) was stored in a liquid nitrogen-containing cell cryopreservation tank to prepare for cloning of a cancer regression antigen gene recognized by CTL. Reference example 2

Preparation of HLA-Cw0102 cDNA

According to the teaching of Nakao et al., Cancer Res. 55: 4248-252 (1995), HLA-Cw0102 cDNA derived from TE8 cells was incorporated into an expression vector pCR3 (manufactured by INVITROGEN) to produce a recombinant plasmid. : Reference example 3

Preparation of TE8 cell cDNA library

Using an mRNA purification system (manufactured by Pharmacia Biotech), separation of the total RNA fraction from TE8 cells and preparation of poly (A) + mRNA using an oligo (dT) column were performed according to the attached protocol. Using Superscript Plasmid System (GIBC0 BRL) from mRNA and preparing cDNA with Not 1 adapter and Sal 1 adapter linked at both ends according to the attached protocol, express this cDNA pSV-SP0RT1 (GIBCO BRL) and ligated to the restriction sites Not 1 and Sal 1 to obtain a recombinant plasmid. This recombinant plasmid was subjected to an electric pulse under the conditions of 25 zF, 200 Ω, and 2.5 kV using a Gene Pulser (manufactured by Bio-Rad) and an E.coli elect-mouth Matsux DH10B / p3 ^™ cell (GIBCO BRL) Transformants with recombinant plasmids selected on LB medium (1% pactotryptone, 0.5% NaCl, pH 7.3) containing ampicillin (50 gZml) did. Reference example 4

Quantitation of Interferon-1

Quantification of interferon (IFN-r) was performed by Enzymimnoassay (ELIS A). 9 Adsorb anti-human IFN-7 mouse monoclonal antibody as the primary antibody on the 6-well mic opening plate, block nonspecific binding with serum albumin, and bind IFN-7 in the sample to the antibody. Was. Then anti-human IF _New as a secondary antibody - to bind the γ © heron poly crawl antibodies after binding the further Peruokishida Ichize labeled anti © heron immunoglobulin donkey antibody, ΤΜ Β Ζ as coloring agent (tetramethyl Rupenjidein) After the reaction, the reaction was stopped by adding an equal amount of 2ΝH2SO4, and the absorbance (450 nm) was measured. This was quantified by comparing it with the value obtained from the standard IFN-α. Example 1

Screening for tumor antigen protein genes

First, recombinant plasmid DNA is recovered from the pool of transformants prepared in Reference Example 3.

Add 100-200 transformants per well to a 96-well U-bottom microplate containing LB medium containing ampicillin (50 / zg / ml), culture, and aliquot 0.3 ml per well. TYGPN medium (F. Ausubel et al., Ed., CURRENT PR0TC0LS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.), transferred to another 96-well U-bottom microplate and incubated at 37 for 48 hours. The remaining microplates of the LB medium were stored frozen. Recombinant plasmid DNA from transformants cultured in TYGPN medium was prepared by microplate-based lysis method (FM Ausubel et al., Ed., CURRENT P0TC0LS IN M0LECULAR BIOLOGY, John Wiley & Sons, Inc.). . Recombinant plasmid DNA recovered by isopropanol precipitation was suspended in a 10 mM Tris, ImM EDTA, pH 7.4 solution containing 50/1 20 ng / ml RNase. Next, the recombinant plasmid DNA prepared above and the recombinant plasmid of the HLA-Cw 0102 cDNA prepared in Reference Example 2 were used in C0S7 cells (Gluzan. Y. Cell, 23: 175-182, 1981). First, transfect the cells simultaneously by the lipofectin method.

C0S7 cells were added at 1 × 10 ⁴ cells / well of a 96-well flat-bottom microplate and cultured for 1 day in 100 μl of RPMI medium containing 10% FCS. 100 ng of the recombinant plasmid of HLA-Cw0102 cDNA prepared in Reference Example 2 was added to about 100 transformants of the recombinant plasmid TE8 cDNA 25 // 1 and the ribofectin reagent (about 100-fold diluted) was added. Ribofectamine, manufactured by GIBC0-BRL) 25; / 1. The obtained mixed solution 501 (fusion suspension of ribosome and recombinant plasmid) was added to the cultured C0S7 cells, and double transfected. Two transfectants were prepared. Transfectants are cultured for 48-72 hours at 37 ° C, the culture medium is removed, and 1 x 10 ⁴ TE8 CTL cells per well are added and 100 1 10% human serum and 50 UZml are added. The cells were cultured in a culture solution containing IL-2 at 37 ° C for 16 to 24 hours. The culture was collected and IFN-y was measured by ELISA.

Next, for the 8 groups in which high IFN-7 production was observed by ELISA, using the pool of approximately 100 to 200 clones / milliliter of the transformants of the corresponding TE8CDNA recombinant plasmids that had been frozen and stored. Screen as follows ο

The transformant pool is cultured for about 6 hours in LB (ampicillin 1 / ml) medium, and the culture is further cultured on a plate of LB agar medium containing ampicillin (δΟ ΐ ΖπιΙ) for 1 day. Transfer 200 colonies from each colony to a total of 8 × 200 colonies in each well of a 96-well microplate, and incubate in the same manner as above under the condition that only one transformant per well is used. Then, recombinant plasmid DNA of TE8 cDNA was prepared. Further, the recombinant plasmid of TE8 cDNA and the recombinant plasmid of HLA-Cw0102 cDNA were double-transfected into C0S7 cells by the same method as described above, and then mixed and cultured with TE8 CTL cells. Reacts IFN-7 in the resulting culture was quantified and positive plasmids were selected. By this operation, two TE8 cell cDNA recombinant plasmid clones reacting with TE8 CTL cells were selected, and named clone 3 and clone 6, respectively. For clones 3 and 6, the same operation was repeated once more to confirm the production of IFN-7 by TE8 CTL cells. Example 2

Nucleotide sequencing of tumor antigen gene

Transformant clones 3 and 6 each having a recombinant plasmid containing the cDNA of the tumor antigen gene of interest were cloned for 14-16 at 37 ° C in LB medium containing 500 ml of ampicillin (50〃1 / ml). After culturing for an hour, the cells were collected by centrifugation. Recombinant plasmid was recovered from the cells according to the PLASMID MAXI kit (QIAGBN). The cDNA is integrated at the site between the SP6 RNA polymerase promoter sequence and the T7 RNA polymerase promoter sequence. Thus, the SP6 Promoter primer and the T7 promoter primer described in the literature (DNA 4: 165, 1985) were synthesized. Next, a didoxyquencing reaction was performed by combining the SP6 Promoter primer or T7 promoter primer with Fluore-dATP Labeling Mix (Pharmacia Biotech) and AutoRead Sequencing Kit (Pharmacia Biotech). The nucleotide sequence of the cDNA was determined from both ends using a fluorescent DNA sequencer (Pharmacia Biotech). The nucleotide sequence of cDNA corresponding to clone 3 was determined to be 532 base pairs in total length, and the nucleotide sequence of cDNA corresponding to clone 6 was determined to be 756 base pairs in total length, as shown in SEQ ID NO: 1 and SEQ ID NO: 4, respectively. 7

Using the Gene Works database, the nucleotide sequences described in SEQ ID NOs: 1 and 4 were searched. The results revealed the following:

Clone 3 As a result of the search, the cDNA nucleotide sequence of clone 3 obtained was the same as that of ubiquitin precursor (P. Kay Lund et al., J. Biol. Chem. Vol. 260, No. 12, pp7609-7613, 1985). It was found to have 100% homology. The deduced amino acid sequence of the obtained cDNA base sequence also had 100% homology with the amino acid sequence of the reported ubiquitin precursor (P. Kay Lund et al., Supra). The cDNA sequence and deduced amino acid sequence are shown in SEQ ID NO: 1.

Clone 6

As a result of the search, the cDNA base sequence of clone 6 obtained had a 3'-UTR (untranslated portion) and a poly (A) tail added to the 3'-end, but the rest was that of histone H1. It had 100% homology with the nucleotide sequence. The deduced amino acid sequence of the obtained cDNA base sequence also had 100% homology with the reported amino acid sequence of histone H1. The cDNA sequence and deduced amino acid sequence are shown in SEQ ID NO: 3. Example 3

Identification of the active fragment of clone 3

1. Preparation of cDNA fragment of clone 3

Fragment 1 (276 bp, 92 amino acids) and Fragment 2 (278 bp, 92 amino acids) were prepared using a TA cloning kit (Invitrogen). The amino acid sequences of fragments 1 and 2 are shown in FIG.

2. Screening of active fragments

The recombinant plasmid DNA prepared above and the recombinant plasmid of HLA-Cw0102 cDNA prepared in Reference Example 2 were cotransfected into C0S7 cells by the method described in Example 2 to produce IFN-α. The amount was determined by ELISA. Figure 2 shows the results obtained. This revealed that fragment 1 was active. The sequence of fragment 1 is shown in SEQ ID NO: 2. Example 4

Identification of active fragment of clone 6

1. Preparation of cDNA fragment of clone 6

Fragment 1 (180 bp, 60 amino acids), Fragment 2 (362 bp, 100 amino acids) and Fragment 3 (281 bp, 93 amino acids) were prepared using a TA cloning kit. The amino acid sequences of fragments 1, 2 and 3 are shown in FIG.

2. Screening of active fragments

The recombinant plasmid DNA prepared above and the HLA-Cw0102 cDNA prepared in Reference Example 2 were co-transfected into C0S7 cells by the method described in Example 2 to produce IFN-α. The amount was determined by ELISA. Figure 4 shows the obtained results. This revealed that fragment 3 was active. The sequence of fragment 3 is shown in SEQ ID NO: 4. Industrial applicability

A medicament for activating antitumor immunity using the tumor antigen protein and the tumor antigen peptide of the present invention; a medicament for treating an autoimmune disease using an antibody against the tumor antigen protein of the present invention; And a medicament containing a DNA encoding a tumor antigen protein and the like, and a method for diagnosing a tumor or an autoimmune disease.

Sequence listing

SEQ ID NO: 1

Array length: 532

Sequence type: nucleic acid

Topology: linear

Sequence type: cDNA

Origin

Organism Name: Homo (Homo sapiens)

Tissue type: Esophageal cancer tissue

Array:

GCCATCTGCG GTGGAGCCGC ACAAA 25

ATG CAG ATT TTC GTG AAA ACC CTT ACG GGG AAG ACC ATC ACC CTC GAG 73 Met Gin He Phe Val Lys Thr Leu Thr Gly Lys Thr lie Thr Leu Glu

1 5 10 15

GTT GAA CCC TCG GAT ACG ATA GAA AAT GTA AAG GCC AAG ATC CAG GAT 121 Val Glu Pro Ser Asp Thr lie Glu Asn Val Lys Ala Lys lie Gin Asp

20 25 30

AAG GAA GGA ATT CCT CCT GAT CAG CAG AGA CTG ATC TTT GCT GGC AAG 169 Lys Glu Gly lie Pro Pro Asp Gin Gin Arg Leu lie Phe Ala Gly Lys

35 40 45

CAG CTG GAA GAT GGA CGT ACT TTG TCT GAC TAC AAT ATT CAA AAG GAG 217 Gin Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn lie Gin Lys Glu

50 55 60

TCT ACT CTT CAT CTT GTG TTG AGA CTT CGT GGT GGT GCT AAG AAA AGG 265 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ala Lys Lys Arg 65 70 75 80

AAG AAG AAG TCT TAC ACC ACT CCC AAG AAG AAT AAG CAC AAG AGA AAG 313 Lys Lys Lys Ser Tyr Thr Thr Pro Lys Lys Asn Lys His Lys Arg Lys

85 90 95

AAG GTT AAG CTG GCT GTC CTG AAA TAT TAT AAG GTG GAT GAG AAT GGC 361 Lys Val Lys Leu Ala Val Leu Lys Tyr Tyr Lys Val Asp Glu Asn Gly

100 105 110

AAA ATT AGT CGC CTT CGT CGA GAG TGC CCT TCT GAT GAA TGT GGT GCT 409 Lys lie Ser Arg Leu Arg Arg Glu Cys Pro Ser Asp Glu Cys Gly Ala

115 120 125

GGG GTG TTT ATG GCA AGT CAC TTT GAC AGA CAT TAT TGT GGC AAA TGT 457 Gly Val Phe Met Ala Ser His Phe Asp Arg His Tyr Cys Gly Lys Cys

130 135 140

TGT CTG ACT TAC TGT TTC AAC AAA CCA GAA GAC AAG 493 Cys Leu Thr Tyr Cys Phe Asn Lys Pro Glu Asp Lys

145 150 155

TAACTGTATG AGTTAATAAA AGACATGAAC TAACAAAAA 532 SEQ ID NO: 2

Sequence length: 276

Sequence type: nucleic acid

Topology: linear

Sequence type: cDNA

Array:

ATG CAG ATT TTC GTG AAA ACC CTT ACG GGG AAG ACC ATC ACC CTC GAG 48 Met Gin He Phe Val Lys Thr Leu Thr Gly Lys Thr lie Thr Leu Glu

1 5 10 15

GTT GAA CCC TCG GAT ACG ATA GAA AAT GTA AAG GCC AAG ATC CAG GAT 96 Val Glu Pro Ser Asp Thr lie Glu Asn Val Lys Ala Lys He Gin Asp

20 25 30

AAG GAA GGA ATT CCT CCT GAT CAG CAG AGA CTG ATC TTT GCT GGC AAG 144 Lys Glu Gly lie Pro Pro Asp Gin Gin Arg Leu lie Phe Ala Gly Lys

35 40 45

CAG CTG GAA GAT GGA CGT ACT TTG TCT GAC TAC AAT ATT CAA AAG GAG 192 Gin Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn lie Gin Lys Glu

50 55 60

TCT ACT CTT CAT CTT GTG TTG AGA CTT CGT GGT GGT GCT AAG AAA AGG 240 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ala Lys Lys Arg 65 70 75 80

AAG AAG AAG TCT TAC ACC ACT CCC AAG AAG AAT AAG 276 Lys Lys Lys Ser Tyr Thr Thr Pro Lys Lys Asn Lys

85 90 SEQ ID NO: 3

Array length: 756

Sequence type: nucleic acid

Topology: linear

Sequence type: cDNA

Origin

Organism: Homo sapiens

Tissue type: Esophageal cancer tissue LZ

OJd sxq J¾ λιο ^ IO BIV sAq sAq Λ sAq sA n jas 130 VVV 03V VOO 300030 OVV VVV X199VV 330 OVV 030 WO 000301

0Π 901 001

BIV BIV sAq sAq usv na s aqj ¾iv λΐθ ^ ΐθ sAq

8 δ 330 VOO OVV OVV OVV 013 VVV ILL 331 300 131 130 100 GOV 300 VVV

96 06 98

UI9 1¾Λ n91 J¾ s jas Λ ⁿ sAq na na sA

908 03V WO 010013 3V 399 OVV 39V 01001330V OVV 310 100 113 VVV

08 9Z 01 99

3Π 3 ^J V usv usv sAq n ^ g 8 dsv JAl ¾iv ¾IV na ¾iv 92 31V 10339V OVV DVV VVV 0V9910 XV9 IVl 300 330 330 130 Oil 000

09 99 OS sXq s people n na ¾ν BIV naq jgg JBA AI J3S Sjy nio sA J9S ¾IV ¾IV

60S VVV VVV 013130100013131119 VOO 30V 103 OVO VVV 13X 330 330

^ 09S

ΙΒΛ BIV sA J¾ 9ii naq nio s ΐ¾Λ OJd OJJ XIO J9S ¾IV s q 8jy

Ϊ9Ϊ 0101309VV 33V 3XV 3130V9 V310100333331000310309VV 103

OS 02

OJd J¾ Ai9 Aio BIV sAq sAq ¾iv ¾1V sAq sAq s Ι¾Λ OJJ BIV s

SU 13303V 100090130 OVV VVV 300030 OVV OVV OVV VIO 1333309VV

91 Οΐ 9 I niO ¾IV OJd OJd BIV ¾1V OJd ¾IV BIV ojj ¾iv J¾ J9S

99 OVO 939133133039 D3013933313003013313013V OVO 03101V

L \ 0VV31311V00001030

^::

Z8TtO //. 6df / XDd Z8SZZ / 86 OAV 115 120 125

AAG AAG CCA GTT GGG GCA GCC AAG AAG CCC AAG AAG GCG GCT GGC GGC 449 Lys Lys Pro Val Gly Ala Ala Lys Lys Pro Lys lys Ala Ala Gly Gly

130 135 140

GCA ACT CCG AAG AAG AGC GCT AAG AAA ACA CCG AAG AAA GCG AAG AAG 497 Ala Thr Pro Lys Lys Ser Ala Lys Lys Thr Pro Lys Lys Ala Lys Lys 145 150 155 160

CCG GCC GCG GCC ACT GTA ACC AAG AAA GTG GCT AAG AGC CCA AAG AAG 545 Pro Ala Ala Ala Thr Val Thr Lys Lys Val Ala Lys Ser Pro Lys Lys

165 170 175

GCC AAG GTT GCG AAG CCC AAG AAA GCT GCC AAA AGT GCT GCT AAG GCT 593 Ala Lys Val Ala Lys Pro Lys Lys Ala Ala Lys Ser Ala Ala Lys Ala

180 185 190

GTG AAG CCC AAG GCC GCT AAG CCC AAG GTT GTC AAG CCT AAG AAG GCG 641 Val Lys Pro Lys Ala Ala Lys Pro Lys Val Val Lys Pro Lys Lys Ala

195 200 205

GCG CCC AAG AAG AAA 656 Ala Pro Lys Lys Lys

210

TAGGCGAACG CCTACTTCTA AAACCCAAAA GGCTCTTTTC AGAGCCACCA CTGATCTCAA 716 TAAAAGAGCT GGATAATTTC TTTAAAAAAA AAAAAAAAAA 756 SEQ ID NO: 4

Array length: 234

Sequence type: nucleic acid Topology: linear

Sequence type: cDNA

Array:

AAG AAG CCC AAG AAG GCG GCT GGC GGC GCA ACT CCG AAG AAG AGC GCT 48 Lys Lys Pro Lys Lys Ala Ala Gly Gly Ala Thr Pro Lys Lys Ser Ala

5 10 15

AAG AAA ACA CCG AAG AAA GCG AAG AAG CCG GCC GCG GCC ACT GTA ACC 96 Lys Lys Thr Pro Lys Lys Ala Lys Lys Pro Ala Ala Ala Thr Val Thr

20 25 30

AAG AAA GTG GCT AAG AGC CCA AAG AAG GCC AAG GTT GCG AAG CCC AAG 144 Lys Lys Val Ala Lys Ser Pro Lys Lys Ala Lys Val Ala Lys Pro Lys

35 40 45

AAA GCT GCC AAA ACT GCT GCT AAG GCT GTG AAG CCC AAG GCC GCT AAG 192 Lys Ala Ala Lys Ser Ala Ala Lys Ala Val Lys Pro Lys Ala Ala Lys

50 55 60

CCC AAG GTT GTC AAG CCT AAG AAG GCG GCG CCC AAG AAG AAA 234 Pro Lys Val Val Lys Pro Lys Lys Ala Ala Pro Lys Lys Lys

65 70 75

Claims

The scope of the claims

1. A partial peptide of a tumor antigen protein having the amino acid sequence of SEQ ID NO: 2, which binds to a major histocompatibility complex (MHC) class I antigen and recognizes a tumor antigen peptide recognized by T cells. The oligonucleotide molecule to be coded.

2. The oligonucleotide molecule according to claim 1, which encodes a tumor antigen peptide comprising at least 7 consecutive amino acid residues in the amino acid sequence of SEQ ID NO: 2.

3. The oligonucleotide molecule according to claim 1 or 2, wherein the nucleotide sequence encoding the tumor antigen protein is the nucleotide sequence of SEQ ID NO: 2.

4. The oligonucleotide according to claim 3, wherein the nucleotide sequence encoding the tumor antigen protein is obtained by substituting, deleting, inserting or adding one or more nucleotides in the nucleotide sequence of SEQ ID NO: 2. Nucleotide molecule.

5. An oligonucleotide molecule that hybridizes under stringent conditions with the oligonucleotide molecule according to any one of claims 1 to 4.

6. An oligonucleotide molecule comprising a coding sequence of a polynucleotide molecule encoding a tumor antigen protein having the amino acid sequence of SEQ ID NO: 2 or a sequence complementary to the base sequence in the 5 ′ non-coding sequence thereof, or a chemical thereof. Decorative body.

7. A tumor antigen peptide encoded by the oligonucleotide according to any one of claims 1 to 5, or a derivative thereof.

8. A medicament comprising the tumor antigen peptide according to claim 7 or a derivative thereof.

9. An antibody against the tumor antigen peptide of claim 7 or a derivative thereof.

10. A medicament comprising the oligonucleotide molecule according to any one of claims 1 to 6.

11. A plasmid having the oligonucleotide molecule according to any one of claims 1 to 6.

12. A transformant transformed with the plasmid of claim 11.

13. A partial peptide of a tumor antigen protein having the amino acid sequence of SEQ ID NO: 4, which is recognized by Τ cells by binding to the major histocompatibility complex (MH C) class I antigen An oligonucleotide molecule encoding

14. The oligonucleotide molecule according to claim 13, which encodes a tumor antigen peptide comprising at least 7 consecutive amino acid residues in the amino acid sequence of SEQ ID NO: 4.

15. The oligonucleotide molecule according to claim 13 or 14, wherein the nucleotide sequence encoding the tumor antigen protein is the nucleotide sequence of SEQ ID NO: 4.

16. The oligo according to claim 15, wherein the nucleotide sequence encoding the tumor antigen protein is obtained by substituting, deleting, inserting or adding one or more nucleotides in the nucleotide sequence of SEQ ID NO: 4. Nucleotide molecule.

17. An oligonucleotide molecule that hybridizes with the oligonucleotide molecule according to any one of claims 13 to 16 under stringent conditions.

1 8. Oligonucleotide molecule consisting of a sequence complementary to the nucleotide sequence in the coding sequence of the polynucleotide molecule encoding the tumor antigen protein having the amino acid sequence of SEQ ID NO: 4 or its 5 'non-coding sequence or its chemical Modifier.

1 9. A tumor antigen peptide encoded by the oligonucleotide according to any one of claims 13 to 17, or a derivative thereof.

20. A medicament comprising the tumor antigen peptide according to claim 19 or a derivative thereof.

2 1. An antibody against the tumor antigen peptide of claim 19 or a derivative thereof.

2 2. A medicine containing the oligonucleotide molecule according to any one of claims 13 to 18

23. A plasmid having the oligonucleotide molecule according to any one of claims 13 to 18.

2 4. A transformant transformed by the plasmid of claim 23.