WO1998022582A1 - Proteine antigenique de regression du cancer chez l'homme - Google Patents

Proteine antigenique de regression du cancer chez l'homme Download PDF

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Publication number
WO1998022582A1
WO1998022582A1 PCT/JP1997/004182 JP9704182W WO9822582A1 WO 1998022582 A1 WO1998022582 A1 WO 1998022582A1 JP 9704182 W JP9704182 W JP 9704182W WO 9822582 A1 WO9822582 A1 WO 9822582A1
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tumor antigen
cells
peptide
sequence
seq
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PCT/JP1997/004182
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English (en)
Japanese (ja)
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Kyogo Itoh
Yasuhisa Imai
Uhi Tho
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Kyogo Itoh
Yasuhisa Imai
Uhi Tho
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Priority to AU49667/97A priority Critical patent/AU4966797A/en
Publication of WO1998022582A1 publication Critical patent/WO1998022582A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • T cells melanoma Toxic T cells
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • 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).
  • MHC class I antigen 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.
  • MAGE Melanoma antigen
  • 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):
  • 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.
  • 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.
  • VLPOVF IRC HLA-A2-restricted antigen peptide
  • 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 g ⁇ 75 (J. Exp. Ed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • STAPPAHGV the peptide derived from the antigen
  • HER-2 / neu 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.
  • 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.
  • tumor antigen proteins that are highly expressed in tumors 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • MH C major histocompatibility complex
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • 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.
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • 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.
  • cytotoxicity of peptide presentation cells it can also be used how to use a peptide-presenting cells labeled with 5 1 C r.
  • 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.
  • 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.
  • the tumor antigen protein for example, B cells of the same patient
  • mass spectrometry was performed. This method is used to determine the sequence.
  • the same amount of gp100 from melanoma cells A tumor antigen peptide derived from offspring Pmell7 has been identified (Science264: 716-719, 1994).
  • a method for determining a gene encoding a tumor antigen protein and further identifying the corresponding tumor antigen peptide 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.
  • 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.
  • 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.
  • 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 c ⁇ L. Med. 176: 1453, 1992, J. Exp. Med. 179: 24, 759, 1994).
  • MHC class I antigens such as HLA-Al, -A0201, -A0205, -All, A31, -A6801, -B7, -B8, -B2705, -37, -CW0401, and -CW0602.
  • 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).
  • 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.
  • 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.
  • the tumor antigen peptide of the present invention or a derivative thereof is encoded by the below-described oligonucleotide of the present invention.
  • 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.
  • 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.
  • an adjuvant those described in the literature (Cl in. Microbiol. Rev. 7: 277-289. 1994) can be applied.
  • 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.
  • 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.
  • 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.
  • the oligonucleotide molecules of the present invention also include "oligonucleotide molecules that hybridize to the oligonucleotide molecules of the present invention under stringent conditions".
  • a DNA molecule that hybridizes to a DNA molecule can be obtained, for example, by the method described in the aforementioned Molecular Cloning.
  • “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.
  • 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.
  • an appropriate vector for example, pBR322, pUC19, pSV • SPORT1, etc.
  • an expression plasmid that replicates and functions in host cells is produced.
  • 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.
  • 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.
  • retrovirus adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, polio virus, and simbis virus.
  • retrovirus adenovirus, adeno-associated virus, vaccinia virus and the like is particularly preferable.
  • ribosome method direct injection of the expression plasmid into the muscle
  • lipofectin method lipofectin method
  • microinjection method calcium phosphate method
  • electoral poration method ribosome method
  • Plasmids having these oligonucleotide nucleotide molecules of the present invention are also included in the scope of the present invention.
  • 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.
  • 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.
  • 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).
  • 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.
  • phosphothioate, phosphorodithioate Derivatives such as alkyl phosphotriesters, alkyl phosphonates and alkyl phosphoamidates ("Ant isense
  • antisense oligonucleotide 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.
  • a preferable length of the antisense oligonucleotide is, for example, 5 to 200 bases.
  • 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.
  • 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.
  • 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.
  • the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
  • CTL cytotoxic T cell
  • 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.
  • 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
  • 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 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
  • RNA 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.
  • oligo (dT) column were performed according to the attached protocol.
  • GIBC0 BRL Superscript Plasmid System
  • GIBCO BRL 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 ⁇ , and 2.5 kV using a Gene Pulser (manufactured by Bio-Rad) and an E.coli elect-mouth Matsux DH10B / p3 TM cell (GIBCO BRL) Transformants with recombinant plasmids selected on LB medium (1% pactotryptone, 0.5% NaCl, pH 7.3) containing ampicillin (50 gZml) did.
  • LB medium 1% pactotryptone, 0.5% NaCl, pH 7.3
  • ampicillin 50 gZml
  • IFN-7 interferon
  • ELIS A Enzymimnoassay
  • recombinant plasmid DNA is recovered from the pool of transformants prepared in Reference Example 3.
  • 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.
  • 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).
  • C0S7 cells were added at 1 ⁇ 10 4 cells / well of a 96-well flat-bottom microplate and cultured for 1 day in 100 ⁇ l 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.
  • Transfectants 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.
  • 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 ( ⁇ ⁇ ⁇ ) 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.
  • 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.
  • 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.
  • the SP6 Promoter primer and the T7 promoter primer described in the literature were synthesized.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 type nucleic acid
  • Organism Name Homo (Homo sapiens)
  • Tissue type Esophageal cancer tissue
  • 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
  • Sequence type nucleic acid
  • 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
  • Sequence type nucleic acid
  • Organism Homo sapiens
  • Tissue type Esophageal cancer tissue LZ
  • 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
  • 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

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Abstract

Cette invention se rapporte à une protéine antigénique tumorale, à un peptide antigénique tumoral correspondant à cette protéine ou similaires, qui peuvent être appliqués à une grande variété de tumeurs, y compris le carcinome spinocellulaire, et qui, même lorsque les tumeurs suceptibles d'être traitées sont limitées, peuvent s'appliquer à une majeure partie des patients souffrant de telles tumeurs ou peuvent s'appliquer à diverses tumeurs, tout en complétant ce procédé avec une immunisation tumorale ou vice-versa. Ces protéines, peptides et similaires peuvent servir à activer l'immunisation contre les tumeurs, à traiter des maladies auto-immunes et à fournir un procédé diagnostic pour les tumeurs et les maladies auto-immunes.
PCT/JP1997/004182 1996-11-21 1997-11-17 Proteine antigenique de regression du cancer chez l'homme WO1998022582A1 (fr)

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AU49667/97A AU4966797A (en) 1996-11-21 1997-11-17 Human cancer regression antigen protein

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JP8/327782 1996-11-21
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01104193A (ja) * 1986-06-10 1989-04-21 Kyowa Hakko Kogyo Co Ltd 新規ポリペプチド
WO1996040917A1 (fr) * 1995-06-07 1996-12-19 Yale University PROTEINES A INTERACTION NuMA ET PROCEDES D'IDENTIFICATION CORRESPONDANTS

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01104193A (ja) * 1986-06-10 1989-04-21 Kyowa Hakko Kogyo Co Ltd 新規ポリペプチド
WO1996040917A1 (fr) * 1995-06-07 1996-12-19 Yale University PROTEINES A INTERACTION NuMA ET PROCEDES D'IDENTIFICATION CORRESPONDANTS

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CANCER RES., 54, (1994), PETER SCHRAML et al., "Identification of Genes Differentially Expressed in Normal Lung and Non-Small Cell Lung Carcinoma Tissue", pages 5236-5240. *
CANCER RES., 55, (1995), K. ITOH et al., "HLA A2601-Restricted CTLs Recognize a Peptide Antigen Expressed on Squamous Cell Carcinoma", pages 4248-4252. *
EUR. J. BIOCHEM., 223, (1994), D. DOENECKE et al., "Role of a Distal Promoter Element in the S-Phase Control of the Human H1.2 Histone Gene Transcription", pages 567-574. *
EUR. J. CELL. BIOL., 49(1), (1989), STEFAN EICK et al., "Human H1 Histones Conserved and Varied Sequence Elements in Two H1 Subtype Genes", pages 110-115. *
EUR. J. IMMUNOL., 21(11), (1991), CLAUDE AURIAULT et al., "Effect Ofubiquitin on Platelet Function: Possible Identify with Platelet Activity Suppressive Lymphokinase (PASL)", pages 2735-2741. *
J. BIOL. CHEM., 260(12), (1985), P. KAY LUND et al., "Nucleotide Sequence Analysis of a cDNA Encoding Human Ubiquitin Reveals that Ubiquitin is Synthesized as a Precursor", pages 7609-7613. *
SCIENCE, 254, (1991), C. TRAVERSARI et al., "A Gene Encoding an Antigen Recognized by Cytolytic T Lymphocytes on a Human Meianoma", pages 1643-1647. *

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