Patent Application
20050043512
The present invention relates to a testis-derived antigenic protein, having an immunity induction activity comparable to a glioma antigen, expressed and exhibiting an immunological response in glioma, which is a malignant brain tumor of humans. The present invention also relates to a gene encoding the abovementioned antigenic protein as well as an immunity induction promoter or suppressor screening method, a glioma detection/diagnosis probe, antitumor agent, etc., that use the glioma antigen and a gene encoding the same.
Though immunotherapy has been anticipated since previously and various efforts have been made, adequate antitumor effects have yet to be demonstrated. Though nonspecific immunotherapy methods have mainly been practiced for immunotherapy of cancers priorly, it has recently become known that with human melanoma, T cells play an important role in tumor rejection in a living body, and since the reporting of the MAGE-1 antigen by a Belgian group in 1991 (Science 254, 1643-7, 1991), efforts are being made to isolate T cell recognizing tumor antigens that can induce cytotoxic T lymphocytes (CTLs) and to determine the MHC class I binding epitope. The present inventors have isolated a CD8+ T cell recognizing antigen by cDNA expression cloning using tumor reactive T cells of melanoma (Proc. Natl. Acad. Sci. USA 91, 3515-3519, 1994; Proc. Natl. Acad. Sci. USA 91, 6458-6462, 1994; J. Exp. Med. 180, 347-352, 1994; J. Immunol. 154, 3961-3968, 1995; Immunologic Res. 16, 313-340, 1997) and have reported that specific immunotherapy using this antigen exhibits antitumor effects for some types of melanoma (Microbiology Immunology 42, 803-813, 1998; Kawakami, Y., P. F. Robbins, R. F. Wang et al. Identification of melanoma antigens by T lymphocytes and their use in the immunotherapy of cancer. In Principle and Practice of Oncology. Update. V. DeVita, S. Hellman, S. A. Rosenberg eds. J. B. Lippincott Co. Philadelphia, p1-20, 1996; Nature Med. 4, 321, 1998). It has also been found that an antigen that is recognized by T cells is a peptide that is bound to an MHC molecule, that the peptide does not have to originate in a cell surface protein, and an intranuclear protein or a cytoplasmic protein may also be recognized as an antigen (Immunity 10, 281-7, 1999).
In 1995, the group of Pfreundschuh of Germany and Old and others of the USA reported SEREX (serological identification of recombinant cDNA expression cloning; Proc. Natl. Acad. Sci. USA 92, 11810-11813, 1995) as a method of detecting cancer antigenic proteins, recognized by IgG antibodies in the sera of cancer patients, that requires neither tumors nor CTLs and can be applied to tumors for which the establishment of a cell line is difficult. Several tumor antigens have been isolated by this method, and since MAGE-1, tyrosinase, and other antigens that induce CTLs are included among the antigens isolated by this method, the method has been indicated as being useful for the detection of antigens that are recognized by cellular immunity. It has also been reported that the above method has enabled isolation of cancer antigens, recognized by patient IgG antibodies, in melanoma, renal cancer, esophageal cancer, colon cancer, lung cancer, etc., (Int. J. Cancer 72, 965-971, 1997; Cancer Res. 58, 1034-1041, 1998; Int. J. Cancer 29, 652-658, 1998; Int. J. Oncol. 14, 703-708, 1999; Cancer Res. 56, 4766-4772, 1996; Hum. Mol. Genet 6, 33-39, 1997). These antigens include CT (cancer-testis) antigens (SSX2/HOM-MEL-40, NY-ESO-1, SCP-1, CT7, etc.), differentiation antigens (galectin-4/NY-CO-27), mutation antigens (p53, etc.), and other important antigens. In particular among these antigens, the HLA-A2 binding epitope has been determined for NY-ESO-1. The antigens isolated by SEREX also include molecules, such as elF-4γ and HER2/neu, which are highly expressed in cancers and these molecules are possibly related to cancerization. Such molecules that are highly expressed in cancers may be applied not only to treatment but has possibilities as diagnostic markers or as markers for estimating recurrence or prognosis.
Meanwhile, the central nervous system has been known as an immune-privileged site and recent studies have shown that stimulated lymphocytes can enter the brain across the blood-brain barrier and react with brain antigens (J. Immunol. 158, 2318-26, 1997; J. Neurosci. 18, 5804-16, 1998). The present inventors have also suggested the possibility of specific immunity induction against tumor antigens in the brain as a new, effective treatment method (Neuro-Oncology 1, S015, 1999). Though presently, image analysis by MRI, etc., and histopathological analysis using biopsy samples are mainly carried out as methods of diagnosing gliomas, which are malignant brain tumors, the genes concerned with the occurrence of glioma are becoming clarified with the progress of molecular cell biology in recent years and the possibilities of genetic diagnosis, in which these genes are analyzed, have also been suggested (Glia. 15, 308-27, 1995).
The SOX gene family is a group of transcription factors defined by the SRY-related HMG box region, which is well conserved and mediates sequence-specific DNA binding (Curr. Opin. Genet. Dev. 7, 338-344, 1997; Nucleic Acids Res. 27, 1409-1420, 1999). With the discovery of the SRY gene (the testes-determining gene on the Y chromosome that determines the male sex) in 1990, a gene group, indicating high homology (>60%) with the SRY HMG box, has been identified by cross hybridization and PCR, and this gene group forms a new subfamily within the HMG box superfamily. SOX genes have been found in a wide range of species from vertebrates, including humans and mice, to invertebrates (fruit fly). At least 20 types of SOX genes have been reported to exist in mice.
The known functions of SOX proteins are diverse and complex. Sry is involved in sex determination (Nature 364, 713-715, 1993), SOX1-3 in lens development (Development 125, 2521-2532, 1998), SOX4 in endocardial ridge development in the heart and pro-B cell proliferation (Nature 380, 711-714, 1996), SOX9 in chondrogenesis and sex determination (Nat. Genet. 22, 85-89, 1999; Cell 79, 1111-1120, 1994) and SOX10 in neural crest cell differentiation (Nat. Genet. 18, 60-64, 1998; Nat. Genet. 18, 171-173, 1998). Such studies provide evidence that SOX genes serve a regulatory role in developmental pathways.
Like the behavior of other SOX genes, the function of the SOX6 protein is considered to be concerned with the restriction of embryonic development and the determination of cell destinies. The mouse SOX6 has been reported to be expressed in the central nervous system during embryogenesis, and though it is expressed until 12.5 days from mating, it has not been detected from the adult brain (Nucleic Acid Res. 23, 3365-3372, 1995). SOX6, SOX5, and SOX9 were found to be expressed at high levels in all cartilaginous parts of the mouse embryo from the early stages of chondrogenesis (Embo. J. 17, 5718-5733, 1998). According to prior reports (Embo. J. 17, 5718-5733, 1998; Gene 265, 157-164, 2001), though the mouse SOX6 is expressed in the fetal brain and primary chondrocytes, such expression is lower than the expression in testis.
Also, two major size classes of SOX6 transcripts have been reported for mice (Proc. Natl. Acad. Sci. U.S.A. 97, 4180-4185, 2000), rats (Biol. Pharm. Bull. 25, 705-709, 2002), and humans (Gene 265, 157-164, 2001). The shorter transcript is approximately 3 kb and the longer transcript is 8 to 9 kb. The difference of these transcripts has been reported to be due to the difference in the respective sizes of the 3′ and 5′ UTRs (untranslated regions) (Embo. J. 17, 5718-5733, 1998), though the possibility of the differences being due to different promoters and different RNA processing cannot be denied.
Presently, even though progresses have been made in the knowledge of the mechanisms of occurrence, diagnostic methods, and treatment methods of cancers, which have become the number one cause of death, much of the advanced cancers cannot be cured in actuality. In particular, malignant brain tumors are disorders that are extremely difficult to cure, and despite progresses in brain surgery and radiation therapy, there have been no effective treatments methods as of yet. The development of new early diagnostic methods and treatment methods is deemed necessary for improvement of the present situation. An object of the present invention is to provide a testis-derived antigenic protein, which has an immunity induction activity comparable to a glioma antigen and is applicable to the diagnosis and treatment of glioma, a gene encoding the same, etc.
In order to achieve the above object, the present inventors carried out diligent research and, by SEREX using a testis-derived cDNA library and glioma patient sera, obtained new testis-derived antigenic proteins, which react only with glioma patient sera, and new testis-derived antigenic proteins, which react with the sera of many glioma patients but hardly react with the sera of healthy individuals. By RT-PCR using glioma antigens, it has been found that the testis-derived antigenic proteins, having immunity induction activities comparable to the glioma antigens, and the DNAs encoding the same are useful for the diagnosis and treatment of glioma and have come to complete the present invention.
The present invention relates to a DNA encoding a protein comprising the amino acid sequence indicated by SEQ ID NO. 2 or 4, or a DNA encoding a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, or added with respect to the amino acid sequence indicated by SEQ ID NO. 2 or 4 and having an immunity induction activity (“1”), a DNA comprising the base sequence indicated by SEQ ID NO. 1 or 3 or a base sequence complementary to the same or a base sequence containing a part or the whole of the same (“2”), a DNA which hybridizes under stringent conditions with the DNA according to “2” and encodes a protein having an immunity induction activity (“3”), and an oligonucleotide or peptide nucleic acid which hybridizes specifically with the DNA according to “3” or with an RNA, corresponding to this DNA, and inhibits the expression of a protein having an immunity induction activity (“4”).
The present invention also relates to a protein comprising the amino acid sequence indicated by SEQ ID NO. 2 or 4, or a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, or added with respect to the amino acid sequence indicated by SEQ ID NO. 2 or 4 and having an immunity induction activity (“5”), a peptide comprising a part of the protein according to “5” and having an immunity induction activity (“6”), a fusion protein or fusion peptide, wherein the protein according to “5” or the peptide according to “6” is bound with a marker protein and/or a peptide tag (“7”), an antibody against the protein according to “5” or the peptide according to “6” (“8”), a recombinant vector containing the DNA according to any of “1” to “4” (“9”), a host cell containing an expression system that can express the protein according to “5” or the peptide according to “6” (“10”), a non-human animal, in which a genetic function encoding the protein according to “5” or the peptide according to “6” is absent from the chromosomes or which overexpresses the protein according to “5” or the peptide according to “6” (“11”), and an immunity induction activity promoter or inhibitor screening method using the protein according to “5” or the peptide according to “6”, a test substance, and T cells and measuring and evaluating the immunity induction activity in the T cells (“12”).
The present invention also relates to a testis-derived antigenic protein having an immunity induction activity comparable to a glioma antigenic protein and being obtained by the steps of: (1) extracting and purifying total RNA from testis tissue, synthesizing cDNA using the purified mRNA, and constructing a λ phage cDNA library by introducing the cDNA into a λ phage vector and infecting Escherichia coli with the vector; (2) diluting the serum of a glioma patient, reacting it with an extract of the Escherichia coli, and removing the reaction products to prepare a reaction serum; (3) reacting the abovementioned λ phage cDNA library with the reaction serum and using a labeled anti-IgG antibody to detect positive clones that react with the antibody in the serum; (4) repeating the screening process several times on the detected positive clones to confirm the antibody reactivity; and (5) isolating an antigen from the positive clones and performing serum screening using the isolated antigen and at least a glioma patient serum and a serum of a healthy individual (“13”), the testis-derived antigenic protein according to “13”, comprising KU-GB-4, KU-GB-3, a SOX family molecule, zinc finger homeobox (zinc finger homeobox 1B), an ADP-ribosylation factor (ADP-ribosylation factor 4-like), or M-phase phosphoprotein 1 (“14”), the testis-derived antigenic protein according to “14”, wherein the SOX family molecule is SOX5, SOX6, or SOX13 (“15”), a glioma detection diagnostic drug containing the whole or a part of the protein according to any of “13” to “15” and/or an antibody binding specifically with the whole or a part of the protein according to any of “13” to “15” (“16”), a glioma diagnosis method characterized in using an antibody binding specifically with the whole or a part of the protein according to any of “13” to “15” (“17”), the glioma diagnosis method according to “17”, wherein a test sample is a serum and ELISA or Western blotting is employed (“18”), the glioma diagnosis method according to “17”, wherein a test sample is a cell tissue and an immunohistochemical analysis method is employed (“19”), a glioma detection/diagnosis probe containing the whole or a part of an antisense chain of a DNA or RNA encoding the protein according to any of “13” to “15” (“20”), an antitumor agent characterized in containing the whole or part of the protein according to any of “13” to “15” and/or an antibody binding specifically with the whole or part of the abovementioned glioma antigen as an effective component (“21”), and a tumor prevention/treatment drug using the antitumor agent according to “21” (“22”).
The present invention furthermore relates to a glioma recognizing antibody specifically recognizing glioma in immunohistochemical analysis (“23”), the glioma recognizing antibody according to “23”, which is an anti-SOX6 antibody (“24”), the glioma recognizing antibody according to “24”, wherein the anti-SOX6 antibody specifically recognizes the HMG box of SOX6 (“25”), a glioma detection diagnostic drug containing the glioma recognizing antibody according to any of “23” to “25” (“26”), a glioma diagnosis method using the glioma recognizing antibody according to any of “23” to “25” (“27”), the glioma diagnosis method according to “27”, wherein a test sample is a serum and ELISA or Western blotting is employed (“28”), the glioma diagnosis method according to “27”, wherein a test sample is a cell tissue and an immunohistochemical analysis method is employed (“29”), and a glioma diagnosis method using the glioma recognizing antibody according to any of “23” to “25” (“30”).
As the present invention's testis-derived antigenic protein, having an immunity induction activity comparable to a glioma antigenic protein, an antigenic protein, obtained by a preparation method comprising the steps of: (1) extracting and purifying total RNA from testis tissue, synthesizing cDNA using the purified mRNA, and constructing a λ phage cDNA library by introducing the cDNA into a λ phage vector and infecting Escherichia coli with the vector; (2) diluting the serum of a glioma patient, reacting it with an extract of the Escherichia coli, and removing the reaction products to prepare a reaction serum; (3) reacting the abovementioned λ phage cDNA library with the reaction serum and using a labeled anti-IgG antibody to detect positive clones that react with the antibody in the serum; (4) repeating the screening process several times on the detected positive clones to confirm the antibody reactivity; and (5) isolating an antigen from the positive clones and performing serum screening using the isolated antigen and at least a glioma patient serum and a serum of a healthy individual; can be cited. Also, it is preferable to use PCR to amplify and determine the sequence of the cDNA insert in the positive clones obtained in the abovementioned step (4) and carry out a homology search using an existing gene database, etc., and in step (5), it is preferable to perform serum screening using, in addition to the glioma patient serum and the healthy individual serum, the sera of other brain disorder patients and other cancer patients.
As specific examples of this testis-derived antigenic protein, one or two or more testis-derived antigenic proteins that are selected from a group of glioma-specific antigens (glioma-specific antigenic genes), which react only with or exhibit a high reactivity to glioma patient sera and include KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6) (GenBank Accession No. NM—006940), SOX6 (GenBank Accession No. NM—033326), SOX13 (GenBank Accession No. NM—005686), etc., zinc finger homeobox (zinc finger homeobox 1B) (GenBank Accession No. NM—014795), an ADP-ribosylation factor (ADP-ribosylation factor 4-like) (GenBank Accession No. NM—033326), etc., and a group of glioma-nonspecific antigenes (glioma-nonspecific antigenic genes), which do not exhibit specificity in reactions with glioma patient sera and include M-phase phosphoprotein 1 (GenBank Accession No. NM—016195), etc., can be cited. Using this testis-derived antigenic protein, dendritic cells, which are powerful antigen-presenting cells, can be expressed in vivo or in vitro and immunity can be induced by the administration of these antigen-expressed dendritic cells.
Of the abovementioned testis-derived antigenic proteins having an immunity induction activity comparable to a glioma antigenic protein, new antigenic proteins and isoforms thereof can be cited as examples of the present invention's protein, with specific examples including the testis-derived antigenic protein KU-GB-3, indicated by SEQ ID NO. 2 in the sequence listing, the testis-derived antigenic protein KU-GB-4, indicated by SEQ ID NO. 4, and proteins, each comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, or added with respect to the amino acid sequence indicated by SEQ ID NO. 2 or 4 and having an immunity induction activity. Here, immunity induction activity refers to the activity of inducing antibody production, cellular immunity, immunologic tolerance, and other immunological reactions, and with regard to such immunity induction activity, a protein that has a T cell induction activity and increases the incidence of precursor cells of cytotoxic T lymphocytes (CTLs) in peripheral blood is especially preferable.
As examples of the present invention's peptide, peptides, comprising a part of any of the abovementioned proteins, having an immunity induction activity, can be cited, and though this peptide is not restricted in particular, a peptide that makes up the recognizing part of an antibody or the recognizing part of a CD4+ T cell and/or CD8+ T cell can be cited as a preferable example. In addition to the abovementioned proteins and peptides that are subjects of the present invention, recombinant proteins and peptides that bind specifically with an antibody that binds specifically with an abovementioned protein or peptide are also included in the present invention, and these may be referred to collectively hereinafter as “the presently concerned glioma antigens.” The origin of the presently concerned glioma antigens is not restricted to humans, and the presently concerned glioma antigens are useful as tumor detection diagnostic drugs and treatment drugs as well as for methods of screening immunity induction activity promoters or inhibitors, as shall be described below.
The DNAs that are subjects of the present invention include DNAs that encode the abovementioned proteins of the present invention, with examples including a DNA encoding the testis-derived antigen protein KU-GB-3, having the amino acid sequence indicated by SEQ ID NO. 2, a DNA encoding the testis-derived antigen protein KU-GB-4, having the amino acid sequence indicated by SEQ ID NO. 4, a DNA encoding a protein, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, or added with respect to the amino acid sequence indicated by SEQ ID NO. 2 or 4 and having an immunity induction activity, and a DNA, comprising the base sequence indicated by SEQ ID NO. 1 or 3 or a base sequence complementary to the same or a base sequence containing a part or the whole of the same. Oligonucleotide sequences, which can be used for amplification, and DNAs and RNAs, which contain a nucleotide sequence that can be hybridized under conditions enabling use as a probe or a marker, are also included in the present invention. These DNAs or RNAs can be used to examine variations of the expression amounts of the presently concerned glioma antigens for the diagnosis of glioma and other cancer disorders related to the expression or function of the presently concerned glioma antigens or the diagnosis of susceptibility to such a disorder.
Also, by using the base sequence indicated by SEQ ID NO. 1 or 3 or a base sequence complementary to the same or a base sequence containing a part or the whole of the same as a probe, performing hybridization under stringent conditions with a DNA library originating from various glioma cells, and isolating the DNA that hybridizes with the abovementioned probe, a DNA, which encodes a protein having an immunity induction activity and intended to be comparable in effect to such antigenic proteins as KU-GB-3 and KU-GB-4, can be obtained. The DNA that is thus obtained also falls within the scope of the present invention. As hybridization conditions for obtaining the present invention's DNA, hybridization at 42 C. and washing treatment at 42° C. by a washing buffer containing 1×SSC and 0.1% SDS can be cited, and hybridization at 65° C. and washing treatment at 65° C. by a washing buffer containing 0.1×SSC and 0.1% SDS can be cited as more preferable conditions. The factors that affect the stringency of hybridization include various factors besides the abovementioned temperature conditions, and those skilled in the art can realize stringency of a level equivalent to the stringency of hybridization conditions given above as examples by appropriate combination of the various factors.
The present invention's oligonucleotide or peptide nucleic acid is not restricted in particular as long as it hybridizes specifically with a DNA having the base sequence indicated by SEQ ID NO. 1 or 3 or a base sequence complementary to the same or a base sequence containing a part or the whole of the same or an RNA corresponding to an abovementioned DNA and can inhibit the expression of a presently concerned glioma antigen (see for example, Bioconjugate Chem. Vol. 5, No. 1, 1994). The abovementioned “peptide nucleic acid” refers to a chemically synthesized nucleic acid analog, which takes the place of a nucleic acid (DNA/RNA), constructed by substituting the pentose/phosphate backbone that is the basic backbone structure of nucleic acid with a polyamide backbone with glycine as a unit, and has a three-dimensional structure similar to a nucleic acid. Such a peptide nucleic acid is preferable in terms of inhibiting the expression of a presently concerned glioma antigen since its bases bind extremely specifically and strongly with a nucleic acid having a complementary base sequence. The abovementioned “oligonucleotide” refers to an oligonucleotide, formed from naturally occurring bases or sugar parts that are bound by a proper phosphodiester bond, etc., or an analog of the same. An oligonucleotide, formed from naturally occurring bases or sugar parts that are bound by a proper phosphodiester bond, etc., refers to a naturally occurring species, a naturally occurring subunit, or a synthetic species formed from a homolog of such a naturally occurring species or naturally occurring subunit. The abovementioned subunit refers to a base-sugar combination bound to an adjacent subunit by a phosphodiester bond or other type of bond.
An analog of an oligonucleotide formed from naturally occurring bases or sugar parts that are bound by a proper phosphodiester bond, etc., refers to an oligonucleotide that functions in a comparable manner to the abovementioned oligonucleotide but has a base that does not occur naturally, for example, a modified base, such as dihydrouridine, inosine, 4-acetylcytidine, 1-methyladenosine, etc., or is increased in stability by chemical modification of the phosphate groups, sugar parts, or the 3′ and 5′ terminals, etc. Examples of methods of chemical modification of the phosphate groups, sugar parts, or the 3′ and 5′ terminals, etc., include but are not limited to the method of substituting one of the oxygen atoms of the phosphodiester group between nucleotides by sulfur and thereby obtaining an oligophosphorothioate, the method of substituting one of the oxygen atoms of the phosphodiester group between nucleotides by —CH3 and thereby obtaining an oligomethylphosphonate, the method of substituting a phosphodiester bond part by another structure that is nonionic and nonchiral, etc.
The abovementioned oligonucleotide may be produced by one skilled in the art by a known synthesis method, for example, by a solid-phase synthesis method using a synthesis device made by Applied Biosystems Inc., etc. Also, other oligonucleotide analogs, such as phosphorothioates and alkylated derivatives may be produced using similar methods (Akira Murakami et al., “Chemical synthesis of functional antisense DNA,” Organic Synthetic Chemistry, 48(3): 180-193, 1990). The present invention's oligonucleotides and peptide nucleic acids that are obtained by such methods can restrain the forming of the presently concerned glioma antigens in animals.
Also, as shall be described in detail below, the abovementioned gene or DNA encoding a presently concerned glioma antigen or a presently concerned glioma antigen, etc., can be used to prepare a fusion peptide or fusion protein, wherein a presently concerned glioma antigen is bound with a marker protein and/or a peptide tag, an antibody against a presently concerned glioma antigen, a recombinant vector containing a DNA encoding a presently concerned glioma antigen, a host cell, containing an expression system that can express a presently concerned glioma antigen, a non-human animal, in which a genetic function encoding a presently concerned glioma antigen is absent from the chromosomes, or a non-human animal, which overexpresses a presently concerned glioma antigen.
The present invention's fusion protein or fusion peptide may be any fusion peptide or fusion protein as long as a presently concerned glioma antigen is bound with a marker protein and/or a peptide tag. Specific examples of marker proteins include but are not limited to priorly known marker proteins, such as alkaline phosphatase, the Fc region of an antibody, HRP, GFP, etc., and specific examples of peptide tags include but are not limited to priorly known peptide tags such as His tags, FLAG tags, S tags, etc. Such a fusion protein can be prepared by a routine procedure and is useful for the purification of an antigen protein, such as KU-GB-3, KU-GB-4, etc., by use of the affinity of Ni-NTA and a His tag, the detection of proteins having T cell induction activity, the quantification of an antibody against an antigen protein, such as KU-GB-3, KU-GB-4, etc., and is also useful as a diagnostic marker for glioma or other type of cancer or a research reagent in the field of the art.
Immune specific antibodies, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, straight-chain antibodies, humanized antibodies, etc., can be cited as specific examples of the present invention's antibody against a presently concerned glioma antigen, and though these can be prepared by routine procedures using the presently concerned glioma antigens mentioned above, monoclonal antibodies are preferable among such antibodies in terms of specificity, and in particular, a monoclonal antibody that specifically recognizes the epitope of KU-GB-3 or KU-GB-4, etc., or a complex of such an epitope and HLA is more preferable. Such monoclonal antibodies and other antibodies are useful not only for the diagnosis of glioma and other cancers and missile therapy and other treatment methods but are also useful for clarifying the initiation mechanisms of glioma and other malignant tumors.
The present invention's antibody is produced using conventional protocols and by administering, to an animal (preferably besides a human), a presently concerned glioma antigen or a fragment thereof that contains an epitope, or cells, on the membrane surface of which is expressed a presently concerned glioma antigen, in particular, a complex of an epitope and HLA. For example for the preparation of a monoclonal antibody, an optional method that provides an antibody produced by a continuous cell culture, such as the hybridoma method (Nature 256, 495-497, 1975), trioma method, human B cell hybridoma method (Immunology Today 4, 72, 1983), and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985), may be used.
For preparation of the abovementioned straight-chain antibody against a presently concerned glioma antigen, a method of preparing a straight-chain antibody (U.S. Pat. No. 4,946,778) may be applied. A transgenic mouse or other mammal can be used to express a humanized antibody, and using this antibody, a clone that expresses the presently concerned glioma antigen can be isolated and identified or the polypeptide can be purified using affinity chromatography. There is a possibility for an antibody against the presently concerned glioma antigens and peptides containing epitopes thereof to be used for the diagnosis and treatment of glioma, etc. Recombinant proteins or peptides to which such antibodies bind specifically are also included as mentioned above among the presently concerned glioma antigens of the present invention.
The present invention's recombinant vector is not restricted in particular as long as it is a vector that contains a DNA encoding a presently concerned glioma antigen, and a vector, containing an expression system that can make a presently concerned glioma antigen be expressed inside a host cell, is preferable. Examples of such a vector include vectors containing an expression system derived from a chromosome, episome, or virus, and to be more specific, include bacterial plasmid derived vectors, yeast plasmid derived vectors, vectors derived from SV40 and other papova viruses, vaccinia viruses, adenoviruses, chicken pox viruses, pseudorabies viruses, and retroviruses, and vectors derived from bacteriophages, transposons, and combinations of these, such as a vector derived from the genetic elements of a cosmid, phagemid, or other plasmid and a bacteriophage. The expression system may contain not only a sequence that causes expression but may also contain a control sequence that regulates the expression.
Host cells, containing an expression system that can express a presently concerned glioma antigen of the present invention, may be prepared by introducing a gene encoding the presently concerned glioma antigen in host cells, and such introduction of a DNA encoding a presently concerned glioma antigen, a vector containing such a DNA, or other expression system mentioned above into host cells can be carried out by methods described in various standard laboratory manuals, such as that by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), that is for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic resin mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection etc. Though as host cells, prokaryotic cells of bacteria, such as Escherichia coli, streptomyces, Bacillus subtilis, streptococcus, staphylococcus, etc., cells of fungi, such as yeast, aspergillus, etc., insect cells, such as Drosophila S2, Spodoptera Sf 9, animal cells, such as L cells, CHO cells, COS cells, HeLa cells, C127 cells, BALB/c3T3 cells (including mutant cell lines lacking dihydrofolic acid reductase, thymidine kinase, etc.), BHK21 cells, HEK293 cells, Bowes malignant melanoma cells, etc., plant cells, etc., can be cited, human cells are preferable.
Also, prokaryotic cells of bacteria, such as Escherichia coli, streptomyces, Bacillus subtilis, streptococcus, staphylococcus, etc., cells of fungi, such as yeast, aspergillus, etc., insect cells, such as Drosophila S2, Spodoptera Sf 9, animal cells, such as L cells, CHO cells, COS cells, HeLa cells, C127 cells, BALB/c3T3 cells (including mutant cell lines lacking dihydrofolic acid reductase, thymidine kinase, etc.), BHK21 cells, HEK293 cells, etc., plant cells, etc., can be cited as examples of the abovementioned host cells. Also, host cells, having HLA expression ability and containing an expression system that can express a presently concerned glioma antigen, can be prepared by introducing a gene encoding the presently concerned glioma antigen by an abovementioned method into host cells having an HLA expression ability or host cells, which do not originally have an HLA expression ability but into which HLAcDNA has been transfected.
The expression system may be any expression system that can make a presently concerned glioma antigen be expressed inside host cells, and may be an expression system derived from chromosomes, episomes, and viruses, such as bacterial plasmid derived vectors, yeast plasmid derived vectors, vectors derived from SV40 and other papova viruses, vaccinia viruses, adenoviruses, adeno-associated viruses, chicken pox viruses, pseudorabies viruses, and retroviruses, and vectors derived from bacteriophages, transposons, and combinations of these, such as a vector derived from the genetic elements of a cosmid, phagemid, or other plasmid and a bacteriophage. This expression system may contain not only a sequence that causes expression but may also contain a control sequence that regulates the expression.
Host cells containing an abovementioned expression system or the cell membranes of such cells or the presently concerned glioma antigens that are obtained by culturing such cells can be used in the present invention's screening method as shall be described later. As a method of obtaining cell membranes, the method of F. Pietri Rouxel et al. (Eur. J. Biochem., 247, 1174-1179, 1997), etc., may be used, and for the recovery and purification of such a presently concerned glioma antigen from a cell culture, known methods, including ammonium sulfate or ethanol precipitation, acidic extraction, anionic or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography, may be used, and high-performance liquid chromatography is preferably used. Particularly in regard to a column to be used in affinity chromatography, a presently concerned glioma antigen can be obtained with a column to which an antibody against the presently concerned glioma antigen is bound, or, in an abovementioned case where a commonly-used peptide tag is added to the presently concerned glioma antigen, with a column to which a substance that has affinity to the peptide tag is bound.
The present invention's non-human animal, in which a genetic function encoding a presently concerned glioma antigen is absent from the chromosomes, refers to a non-human animal, which has lost the function of expressing the presently concerned glioma antigen by inactivation by destruction, loss, substitution or other genetic mutation of a part or the whole of a gene encoding the presently concerned glioma antigen on the chromosomes. The present invention's non-human animal, which overexpresses a presently concerned glioma antigen refers to a non-human animal that produces the presently concerned glioma antigen in large amounts in comparison to a wild type non-human animal. Examples of the abovementioned non-human animal include but are not limited to rodents, such as mice, rats, etc., and other non-human animals.
Homozygous non-human animals that are born according to Mendel's law include both a type lacking or overexpressing a presently concerned glioma antigen and a wild type littermate, and precise comparative experiments at an individual level can be conducted by simultaneously using the type lacking or overexpressing the presently concerned glioma antigen and wild type littermate. It is thus preferable to use, along with the non-human animal, in which the genetic function encoding a presently concerned glioma antigen is absent from the chromosomes or is overexpressed, a wild type non-human animal, that is an animal of the same species or an animal of the same brood in carrying out, for example, the present invention's screening to be described below. Methods of preparing a non-human animal, in which a genetic function encoding a presently concerned glioma antigen is absent from the chromosomes or is overexpressed, shall now be described using a knockout mouse or transgenic mouse for a presently concerned glioma antigen as an example.
For example, to prepare mice, in which a genetic function encoding a presently concerned glioma antigen is absent from the chromosomes, that is, knockout mice for a presently concerned glioma antigen, a gene encoding the presently concerned glioma antigen is screened using gene fragments obtained by PCR or other method from a mouse gene library, and the screened gene encoding the presently concerned glioma antigen is subcloned using a viral vector, etc., and specified by DNA sequencing. The whole or part of this clone's gene encoding the presently concerned glioma antigen is then substituted in a pMC1 neo gene cassette, etc., and a targeting vector is prepared by introducing a gene, such as a diphtheria toxin A fragment (DT-A) gene or a herpes simplex virus thymidine kinase (HSV-tk) gene, into the 3′-terminal side.
The prepared targeting vector is linearized and then introduced into ES cells by a method such as electroporation. The ES cells are then homologously recombined., and from among the homologous recombinants, ES cells, in which homologous recombination was caused by G418, ganciclovir (GANC) or other such antibiotics, are selected. It is preferable to confirm by Southern blotting etc., whether these selected ES cells are the desired recombinants. Clones of the confirmed ES cells are then microinjected into mouse blastocysts, and the blastocysts are transplanted into a recipient mouse to generate chimeric mice. Heterozygous mice can be obtained by intercrossing the chimeric mice with wild-type mice, and knockout mice for the presently concerned glioma antigen can be obtained by intercrossing the heterozygous mice. In addition, as a method for confirming whether knockout mice for the presently concerned glioma antigen have been obtained, for example, RNA is isolated from mice obtained by the above-described method and examined by Northern blotting, etc., or the expression of the presently concerned glioma antigen in the mice is examined by Western blotting, etc.
Also transgenic mice for the present invention's glioma antigen can be prepared by constructing a transgene by fusing cDNA, encoding a presently concerned glioma antigen, with a chicken β-actin, mouse neurofilament, or SV40 promoter etc., and a rabbit β-globin or SV40 polyA or intron etc., microinjecting the transgene into the pronucleus of mouse fertilized egg, culturing and then transplanting the obtained egg cells into the oviduct of a recipient mouse, and thereafter tending the recipient animal and selecting infant mice having the abovementioned cDNA from among the infant mice that are born. The selection of infant mice with the cDNA can be carried out by extracting crude DNA from the tails, etc., of the mice and performing dot hybridization using the introduced gene, encoding the presently concerned glioma antigen, as a probe or performing PCR using a specific primer, etc.
The present invention's method for screening an immunity induction activity promoter or inhibitor is not restricted in particular as long as it uses a presently concerned glioma antigen, a test substance, and T cells to measure and evaluate immunity induction activity in T cells, and a method of using a test substance, a presently concerned glioma antigen, and T cells to measure and evaluate immunity induction activity in the abovementioned T cells, a method of using a test substance, cell membranes or cells expressing a presently concerned glioma antigen, and T cells to measure and evaluate immunity induction activity in the abovementioned T cells, a method of using host cells, which express a presently concerned glioma antigen and have been transfected by both an HLA-expressing vector and a vector expressing a peptide to be tested, and T cells to measure and evaluate immunity induction activity in the abovementioned T cells, a method of using host cells, which express a presently concerned glioma antigen, have an HLA expressing ability, and have been transfected by a vector expressing a polypeptide to be tested, and T cells to measure and evaluate immunity induction activity in the abovementioned T cells, a method of administering a test substance to the above-described knockout mouse, transgenic mouse, or other non-human animal to measure and evaluate the immunity induction activity of the T cells in the non-human animal, etc., can be cited as examples. Specific examples of the abovementioned cell membranes or cells include cells, such as primary cultured cells obtained from a non-human animal, which overexpress the abovementioned gene encoding a presently concerned glioma antigen, or a wild type non-human animal, etc., host cells, containing the abovementioned expression system that can express a presently concerned glioma antigen, and cell membranes of such cells, etc. As a method of putting such cell membranes or cells into contact with a test substance, a method of culturing cell membranes or cells expressing a presently concerned glioma antigen in vitro, in the presence of the test substance, and then putting them into contact with T cells, etc., can be cited as examples. As a method of measuring and evaluating immunity induction activity in T cells, a method of evaluating the amount of IFNγ, which is released from the T cells to a medium, as an index can be cited as a specific example. An immunity induction activity promoter obtained by the above-described screening method can be used for treatment of a patient requiring the promotion of immunity induction activity, etc., and an immunity induction activity inhibitor obtained by the above-described screening method can be used for treatment of a patient requiring the inhibition of immunity induction activity, etc.
The present invention's glioma detection diagnostic drug is not restricted in particular as long as it is a reagent containing a testis-derived antigen protein or part thereof that has an immunity induction activity comparable to the present invention's glioma antigen protein. An antigen recognition part of a glioma antigen, etc., can be cited as an example of a part of an antigen protein, and in a case where two or more types of antigens are to be used in combination, the antigens are preferably selected from among KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6) (GenBank Accession No. NM—006940), SOX6 (GenBank Accession No. NM—033326), SOX13 (GenBank Accession No. NM—005686), etc., zinc finger homeobox (GenBank Accession No. NM—014795), ADP-ribosylation factors (GenBank Accession No. NM—033326) and other proteins of the glioma-specific antigen group or M-phase phosphoprotein 1 and other proteins of the glioma-nonspecific antigen group. Reagents, containing a monoclonal antibody or other antibody that specifically binds to the abovementioned testis-derived antigen protein or part thereof that has an immunity induction activity comparable to the present invention's glioma antigen protein, may also be used as the present invention's glioma detection diagnostic drug, and even with these antibodies, in a case where two or more such antibodies are to be used in combination, selection is preferably made from among antibodies against KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6), SOX6, SOX13, etc., zinc finger homeobox, ADP-ribosylation factors and other proteins of the glioma-specific antigen group or M-phase phosphoprotein 1 and other proteins of the glioma-nonspecific antigen group. Examples of methods of diagnosing glioma or other cancers using the present invention's glioma detection diagnostic drug include a method of using a labeled glioma antigen and making it react with an IgG antibody in a serum obtained from a test subject to examine whether or not an immune reaction is seen, a method of administering an antibody, which is fluorescence labeled or labeled with iron, etc., and is an antibody against the abovementioned testis-derived antigen protein or part thereof that has an immunity induction activity comparable to the present invention's glioma antigen protein, into blood and carrying out image diagnosis of glioma or other cancer with unclear boundaries, etc.
The present invention's glioma detection/diagnosis probe is not restricted in particular as long as it is a reagent containing an antisense chain of a DNA or RNA encoding the abovementioned testis-derived antigen protein or part thereof that has an immunity induction activity comparable to the present invention's glioma antigen protein. Specific examples include reagents containing a part or the whole of an antisense chain of a DNA or RNA encoding one or two or more types of glioma antigen selected from among KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6), SOX6, SOX13, etc., zinc finger homeobox, ADP-ribosylation factors and other proteins of the glioma-specific antigen group or M-phase phosphoprotein 1 and other proteins of the glioma-nonspecific antigen group, and a DNA or RNA of 20 bp or more is preferable, and in a case where two or more probes are to be used in combination, the probes are preferably selected from among the abovementioned antisense chains of DNA or RNA encoding one or two or more types of antigen proteins selected from among KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6), SOX6, SOX13, etc., zinc finger homeobox, ADP-ribosylation factors and other proteins of the glioma-specific antigen group or M-phase phosphoprotein 1 and other proteins of the glioma-nonspecific antigen group. As an example of a glioma detection/diagnosis method using the present invention's glioma detection/diagnosis probe, a method of using a labeled antisense chain to detect the mRNA of the abovementioned testis-derived antigen protein or part thereof that has an immunity induction activity comparable to the present invention's glioma antigen protein obtained from a test subject, can be cited. Specific examples of the test subject used in this detection/diagnosis method include but are not restricted to genome DNA, RNA, and cDNA that can be obtained from cells of a subject, for example, cells in blood, urine, saliva, tissue, and other samples obtained by biopsy.
As the present invention's antitumor agent, a preparation containing, as an effective component thereof, the whole or a part of one type or two or more types of antigen protein obtained by the abovementioned method of preparing the present invention's testis-derived antigen protein having an immunity induction activity comparable to a glioma antigen protein, can be used. An antigen recognizing part, etc., of a glioma antigen can be cited as an example of a part of an antigen protein, and in a case where two or more antigens are to be used in combination, the antigens are preferably selected from among KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6), SOX6, SOX13, etc., zinc finger homeobox, ADP-ribosylation factors and other proteins of the glioma-specific antigen group or M-phase phosphoprotein 1 and other proteins of the glioma-nonspecific antigen group. Also as the present invention's antitumor agent, a preparation, containing, as an effective component thereof, an antibody, such as a monoclonal antibody, polyclonal antibody, chimeric antibody, straight-chain antibody, humanized antibody, etc., that binds specifically with the whole or a part of the abovementioned glioma antigen, can be used, and even with such antibodies, when two or more such antibodies are to be used in combination, selection is preferably made from among antibodies against KU-GB-3, KU-GB-4, SOX family molecules, such as SOX5 (sex determining region Y BOX6), SOX6, SOX13, etc., zinc finger homeobox, ADP-ribosylation factors and other proteins of the glioma-specific antigen group or M-phase phosphoprotein 1 and other proteins of the glioma-nonspecific antigen group. In using the present invention's antitumor agent, various commonly-used formulation components for drug preparation that are pharmaceutically allowed, such as carriers, binders, stabilizers, fillers, diluents, pH buffers, disintegrators, dissolution agents, solubilizing agents, isotonic agents, etc., may be added. These treatment drugs and biophylactic enhancement drugs may be administered orally or parenterally. That is, the drugs may be administered by a commonly employed form of administration, for example, parenteral administration by injection of a preparation in the form of a solution, emulsion, suspension, etc., or oral administration of a preparation in the form of a powder, granules, capsule, syrup, suspension, etc. In the case of oral administration, the antitumor agent is preferably prepared in an abovementioned liposome sealed/embedded form. The present invention's treatment method for glioma or other cancer disorder is not restricted in particular as long as it is a treatment method that uses the abovementioned antitumor agent, and antitumor effects due to increases in vivo activation of T cell induction can be anticipated by the administration of this antitumor agent orally or by intravenous, intracutaneous, or subcutaneous injection, etc. Also, T cells can be stimulated in vitro and induced to become activated T cells using the present invention's antitumor agent, and for example, when peripheral blood lymphocytes or tumor-infiltrated lymphocytes are stimulated by IL-2 and the present invention's antitumor agent, tumor reactive activated T cells are induced and these activated T cells can be used effectively in adoptive immunotherapy. The present invention's monoclonal antibodies and other antibodies can also be used to perform a genetic treatment, wherein a gene is selectively introduced into glioma, a missile therapy, wherein an antitumor agent is made to act selectively on glioma, etc.
Though the present invention shall now be described more specifically by way of examples, the technical scope of the present invention is not limited to these examples.
[Cell Lines and Tissues]
Human glioma cell lines (GI-1, T98G, U87MG, U251, A172), a human malignant melanoma cell line (888MEL), lung cancer cell lines (LU99, EBC1), an esophageal cancer cell line (TE10), a pancreatic cancer cell line (PK1), a bladder cancer cell line (KU7), a prostatic cancer cell line (PC3), a breast cancer cell line (MDA231), a leukemia cell line (MOLT4), and fibroblasts were respectively cultured in RPMI, containing 10% bovine fetus serum along with penicillin (100 IU/ml) and streptomycin (100 μg/ml). As human glioma tissues [GB (glioblast) 13, GB17, GB16, GB4], parts of lesions extracted by surgery were used. RNAs of normal tissues (brain, heart, lung, stomach, small intestine, large intestine, liver, spleen, kidney, testis, placenta, muscle, fetal brain) were purchased from Clontech Laboratories Inc.
[Construction of cDNA Library]
Total RNA of adult human testis was purchased from Clontech Laboratories Inc. Poly(A)+ RNA was purified twice using latex beads coated with oligodeoxythymidylic acid (Oligotex-dT30 super; Takara Shuzo Co., Ltd.). By reverse transcription using 5 μg of the poly(A)+ RNA and the ZAP-cDNA Synthesis Kit (Stratagene Inc.), a testis cDNA library was constructed. Upon insertion of cDNA fragments into the bacteriophage expression vector, λAzapII (Stratagene Inc.), 3 ×106 primary recombinants were obtained as the library.
[Screening of Test is cDNA Library by Serum]
The cDNA library constructed as described above was sown at an amount of 1×104 clones in a 150 mm NZY agarose plate and incubated at 42° C. for 4 hours, followed by further incubation at 37° C. for 4 hours. Recombinant proteins, expressed and induced by 10 mM of IPTG on Escherichia coli (XL1-Blue), were transferred onto nitrocellulose filters (Hybond-c; Amersham, Buckinghamshire, England), and after removing the adsorbed bacteria and phages by washing the filters with TBS (10 mM of Tris-HCl, 150 mM of NaCl; pH 7.5), containing 0.05% of TWIN 20, nonspecific reactions were inhibited with TBS containing 5% skim milk. The filters were then reacted at 37° C. for 4 hours with patient sera diluted by 1:400. As patient sera, sera were sampled respectively from the twelve glioma patients shown in Table 1 and mixtures, each of sera of four glioma patients [(mixture of patient sera G1, G3, G5, and G8), (mixture of patient sera G20, G21, G22, and G27), (mixture of patient sera G18, G23, G24, and G28)], were used. These patient sera were stored at −80° C., and immediately prior to use, each serum was diluted by 1:5 with a TBS solution containing 5% skim milk. In order to priorly eliminate antibodies that react with bacteria, each serum was mixed at a ratio of 1:2 with a lysate of lysed Escherichia coli and reacted at 4° C. for 8 hours, the resulting supernatant was diluted by 1:400 in the final stage, and this diluted supernatant was used as the serum.
The sera that were thus processed were then reacted at 37° C. for 4 hours with the recombinant proteins that were plotted on the nitrocellulose filters, and the recombinant proteins that reacted with the antibodies in sera were incubated with alkaline phosphatase-labeled goat antihuman IgG (Fc) antibody (Cappel Inc.). Enzymatic detection of bound secondary antibodies was then carried out using nitro blue tetrazolium (Boehringer Mannheim GmbH) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co.). Positive clones corresponding to the parts of positive color reaction were then sampled from the abovementioned 150 mm NZY agarose plate and dissolved in SM buffer solution (100 mM of NaCl, 10 mM of MgSO4, 50 mM of Tris-HCl, 0.01% of gelatin; pH 7.5). This screening process was repeated several times to confirm the antibody reactivity of the positive clones. 58 positive clones were isolated by screening from 5×105 phage clones per one patient's serum.
[Homology Search of Isolated Antigen Gene]
The cDNA inserts that were incorporated in the 58 phages obtained as described above were then amplified by PCR using the ExTaq kit (Takara Shuzo Co., Ltd.) and the respective base sequences were determined. ExTaq (Takara Shuzo Co., Ltd.) was used as the reaction enzyme, T3 (5′-AATTAACCCTCACTAAAGGG-3′; SEQ ID NO. 5) was used as the sense primer, and T7 (5′-GTAATACGACTCACTATAGGGC-3′; SEQ ID NO. 6) was used as the antisense primer. In regard to reaction conditions, a thermal cycler (Perkin Elmer) was used to perform denaturing at 94° C. for 5 minutes just at first and then to repeat a cycle of heat denaturing at 94° C. for 1 minute, annealing at 55° C. for 1 minute, and extension at 72° C. for 2 minutes 35 times. Lastly, extension at 72° C. was carried out for 7 minutes. The PCR products obtained were subject to DNA sequencing using the Big Dye DNA Sequencing Kit (ABI) and the ABI310 Auto Sequencer and the base sequences at the 5′ side were determined across a length of approximately 300 to 500 bp. The DNA thus determined were compared with the genetic information registered in the genetic databases of the National Center for Biotechnology Information or the EST databases to search for homologies with known genes. As a result, it was found that the abovementioned 58 positive clones were of fifteen types of antigen cDNA consisting of eleven types of cDNAs encoding known proteins and four types of cDNA encoding new proteins. The numbers of types of the fifteen types of antigen cDNA are shown according to patient in Table 1.
[Serum Screening]
In order to examine application to serum diagnosis of glioma, screening was performed using the respective clones that express the isolated fifteen types of antigens and using 1:100 diluted sera of 29 glioma patients (12 glioblastoma patients, 7 anaplastic astrocytoma patients, 4 anaplastic oligoastrocytoma patients, 4 astrocytoma patients, 2 oligoastrocytoma patients), 14 other brain disorder patients (2 malignant lymphoma patients, 1 brain abscess patient, 1 subarachnoid hemorrhage patient, 2 Parkinson's disease patients, 4 meningioma patients, 1 neurocytoma patient, 1 craniopharyngioma patient, 2 metastatic brain tumor patients), and 37 healthy individuals and the sera were examined to see whether or not IgG antibodies against the respective isolated glioma antigens can be detected. As a result, six types of glioma specific antigens (glioma specific antigen genes), which react only with glioma patient sera or react with many of the glioma patient sera but hardly undergo an antibody reaction with the sera of healthy individuals, and one type of glioma nonspecific antigen (glioma nonspecific antigen gene), for which specificity was not found for reaction with glioma patient sera but which does react with glioma patient sera, were obtained. As shown in Table 2, the six types of glioma specific antigens (glioma specific antigen genes), including the new antigen, KU-GB-4, were KU-GB-3, SOX5 (sex determining region Y BOX6), SOX6, zinc finger homeobox, and an ADP ribosylation factor. Also, as shown in Table 3, the one type of glioma nonspecific antigen (glioma nonspecific antigen gene) was M-phase phosphoprotein 1. Though the abovementioned M-phase phosphoprotein 1 did not exhibit specificity in reactions with sera, the six types of glioma specific antigens were found to react specifically with glioma patient sera and are thus considered to be useful as a serum diagnostic drugs or treatment drugs for glioma.
Also as shown in Table 2, screening was performed using separately isolated clones expressing SOX13 and using 1:100 diluted sera of 29 glioma patients (12 glioblastoma patients, 7 anaplastic astrocytoma patients, 4 anaplastic oligoastrocytoma patients, 4 astrocytoma patients, 2 oligoastrocytoma patients) to examine whether or not IgG antibodies against the respective isolated glioma antigens can be detected in these sera. As a result, reactions were detected with exactly the same patient sera as those reacting with SOX5 and SOX6. As with SOX5 and SOX6, SOX13 is a molecule belonging to the SOX D group of the SOX family, and these SOX D group molecules are considered to be useful as serum diagnostic drugs or treatment drugs for glioma.
[Determination of the Base Sequences and Amino Acid Sequences of the Antigens]
As a result of examining the base sequences of the isolated clones using an automatic DNA sequencer (ALF Express; Pharmacia Biotech Inc.), the KU-GB-3 gene was found to comprise a base sequence (SEQ ID NO. 1) with a total length of 2898 bp containing a gene encoding a sequence of 865 amino acids (SEQ ID NO. 2), and the KU-GB-4 gene was found to comprise a base sequence (SEQ ID NO. 3) with a total length of 1748 bp containing a gene encoding a sequence of 399 amino acids (SEQ ID NO. 4). Though the KU-GB-3 gene had been registered as a placenta-derived cDNA in GenBank (Accession No. AK001973), it was not known as a glioma antigen.
[RT-PCR Method for Detecting the Expression of Glioma Antigens]
The expression specificities of the genes of the glioma antigens KU-GB-3, KU-GB-4, SOX5, SOX6, zinc finger homeobox, and M-phase phosphoprotein 1 in normal tissues, glioma cell lines and tissues, and other tumor cell lines were examined by the RT-PCR method. 5 μRg each of RNAs derived from the normal tissues of brain, heart, lung, stomach, large intestine, liver, spleen, kidney, testis, placenta, muscle, fetal brain, etc., (all purchased from Clontech Laboratories Inc.), four types of glioma tissues (GB13, GB17, GB16, and GB4), and four types of glioma cell lines (GI-1, U87MG, T98G, U251, and A172) and an RNA derived from cultured fibroblasts were used along with avian myeloblast virus reverse transcriptase (Takara Co., Ltd.) and oligo (dT) as the primer to prepare a cDNA panel to serve as a mold for RT-PCR at a total reaction amount of 200 μl at 42° C. For the RT-PCR method for detection of expression of the KU-GB-4 gene, 5 μg each of RNAs derived from a malignant melanoma cell line (888MEL), lung cancer cell lines (LU99, EBC1), an esophageal cancer cell line (TE10), a pancreatic cancer cell line (PK1), a bladder cancer cell line (KU7), a prostatic cancer cell line (PC3), a breast cancer cell line (MDA231), and a leukemia cell line (MOLT4) were used.
5′-TCAAGGAACTGGCATACCCTGTCT-3′ (SEQ ID NO. 7: P1) was used as the sense primer and 5′-GGTACTCGACAAGCACTGACAATC-3′ (SEQ ID NO. 8: P2) was used as the antisense primer for detection of KU-GB-3, 5′-CAAACTCTGGGAGGAAGACC-3′ (SEQ ID NO. 9: P3) was used as the sense primer and 5′-AGTTGGCTGAGAAGGACAAG-3′ (SEQ ID NO. 10: P4) was used as the antisense primer for detection of KU-GB-4, 5′-GCAACAGGAGGAAGGAAAT-3′ (SEQ ID NO. 11: P5) was used as the sense primer and 5′-GACAAGCCAACTGATAAGGGTC-3′ (SEQ ID NO. 12: P6) was used as the antisense primer for detection of SOX5, 5′-CGCGCTTTGAGAATTTGGGGCC-3′ (SEQ ID NO. 13: P7) was used as the sense primer and 5′-TCTTTGTTGGGGAGGGGGGTGA-3′ (SEQ ID NO. 14: P8) was used as the antisense primer for detection of SOX6, 5′-TGGAGATCACTCCATGGACGATAG-3′ (SEQ ID NO. 15: P9) was used as the sense primer and 5′-CCTGTTCTTTGAAGCACCCATGT-3′ (SEQ ID NO. 16: P10) was used as the antisense primer for detection of zinc finger homeobox, and 5′-AACGAGCCAAACGGAA-3′ (SEQ ID NO. 17: P11) was used as the sense primer and 5′-GTATCCATCCCCTCAAGC-3′ (SEQ ID NO. 18: P12) was used as the antisense primer for detection of M-phase phosphoprotein 1. For the detection of β-actin (551 bp) as the control, 5′-GTCGACAACGGCTCCGGCATGTGCA-3′ (SEQ ID NO. 19: P13) was used as the sense primer and 5′-GGATCTTCATGAGGTAGTCAGTCAG-3′ (SEQ ID NO. 20: P14) was used as the antisense primer. Using these PCR primers and using ExTaq (Takara Co., Ltd.) as the reaction enzyme, a cycle of 1 minute of heat denaturing at 94° C., followed by annealing and then 30 seconds of extension reaction at 72° C., was repeated 30 times (28 times in the case of KU-GB-3) using a thermal cycler (Perkin-Elmer). The annealing temperature was set to the Tm value of each primer [59° C. for KU-GB-3, 62° C. for KU-GB-4, 63° C. for SOX5, 61° C. for SOX6, 62° C. for zinc finger homeobox, 62° C. for M-phase phosphoprotein 1, and 68° C. for β-actin, serving as the control] and was carried out for 30 seconds. The PCR products obtained were subject to agarose gel electrophoresis (2.0%), stained with ethidium bromide (EtBr), and then bands were detected by irradiation of ultraviolet rays of 254 nm.
The results of the above are shown in
The above results show that the genes for KU-GB-3, KU-GB-4, SOX5, SOX6, zinc finger homeobox, and M-phase phosphoprotein 1 are useful as genetic diagnostic drugs and treatment drugs for cancers, including glioma. In regard to expression in testis, since this tissue is isolated from the immune system, it is considered that there is hardly any possibility of testis disorders occurring when the KU-GB-3, KU-GB-4, SOX5, SOX6, zinc finger homeobox, or M-phase phosphoprotein 1 antigen is actually used in immunotherapy, etc.
The characteristics of SOX6, which was identified by SEREX as being a testis-derived human glioma antigen, were examined in more detail.
[Preparation of Recombinant SOX6 HMG Box Protein]
The cDNA of the SOX6 HMG box was subcloned in pET32a plasmids (Novagen) and the protein was expressed in the Escherichia coli, BL21 (DE3) Lys S (Novagen). The recombinant SOX6 was purified using the affinity His, HiTrap Chelating (Amersham Pharmacia).
[Western Blot Analysis for Evaluating the Serologic Reaction to the SOX6 HMG Box]
The purified recombinant SOX6 HMG box was mixed with an equal volume of sodium dodecyl sulfate (SDS)—sample buffer, composed of 2% SDS, 10% β-mercaptoethanol, 10% glycerol, 1 mM EDTA, 40 mM Tris, and 240 mM glycine at pH 8.5, and boiled for 3 minutes. The same amount of total protein (10 μg) was loaded in respective lanes of SDS-polyacrylamide gels (10%). After electrophoresis, the gels were transferred to a nitrocellulose sheets (Hybond C+; Amersham Pharmacia) by electroblotting. After blocking with 5% skim milk for 2 hours, the sheets were incubated with 1:100 diluted sera from glioma patients for 1 hour at room temperature. After washing, the blots were incubated with 1:2000 diluted alkaline phosphatase-labeled goat antihuman IgG (Fc) antibody (Cappel Inc.). Enzymatic detection of bound secondary antibodies was then carried out using nitro blue tetrazolium (Boehringer Mannheim GmbH) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co.).
[ELISA for Examining the Serologic Reaction to the SOX6 HMG Box]
100 ng per well of the purified recombinant SOX6 HMG box was adsorbed to the wells of a 96-well plate overnight. Control wells were coated with purified recombinant protein pET 32a plasmid. After washing and blocking the wells with 2% BSA/PBS, 100 μl of diluted human serum was added and incubation at 4° C. was carried out for 1 hour. Examinations were carried out with the sera being diluted in the range of 1:100 to 1:50,000. The wells were washed and incubated for 1 hour with a peroxidase-conjugated antibody to human IgG, and this was followed by reaction with a TMB substrate (Dako) and measurement of the optical density at 450 nm.
[Quantitative RT-PCR and Northern Blot]
Using SYBR green, which is a fluorescent dye, and the ABI Prism 7700 Sequence Detection System (Perkin-Elmer, Inc.), quantitative RT-PCR analysis was carried out by a method described in literature (Nat. Med. 4, 1329-1333, 1998). The thermal cycler parameters were set to 10 minutes at 95° C., 50 cycles of denaturation at 95° C. for 30 seconds, annealing at 60° C. for 1 minute, and extension at 72° C. for 1 minute. The relative expression amount of SOX6 was computed by the following procedure. That is, the β-actin of each tissue was used for standardization and the (CT) (threshold cycle value) of each sample was divided by the CT value of normal brain. When the fluorescence signal increases to or above the background threshold level, the CT value is defined as the number of actual PCR cycles and indicates the number of copies of the target gene.
For Northern blot analysis, 10 μg of total RNA was fractionated by electrophoresis in a 1% formaldehyde agarose gel and transferred to a nylon membrane (Hybond-XL; Amersham Pharmacia Inc.). Radioisotope-labeled cDNA fragments were prepared using the High Prime DNA Labeling Kit (Boehringer Mannheim GmbH). Prehybridization was performed at 65° C. for 30 minutes using the Quick Hyb solution (Stratagene Inc.), and hybridization with the probes was carried out overnight at 65° C. The membrane was then washed twice for 15 minutes each at room temperature with 2×SSC in 0.1% SDS, followed by an additional wash for 30 minutes at 60° C. in 0.1×SSC and 0.1% SDS. Radioactive signals were detected using the BAS5000 (Bio-Rad Inc.).
[Transfection of SOX6 into 293T Cells]
Full-length SOX6 cDNA was subcloned in the mammalian expression vector, pcDNA3myc (Invitrogen Inc.). The linearized cDNA was genetically introduced into a 293T cell line using Lipofectamine PLUS reagent (Life Technologies Inc.).
[Isolation of Nuclear Extracts]
Normal brain tissue purchased from Clontech Laboratories Inc., glioma tissues, and 293T cells were homogenized in 0.25 M sucrose and centrifuged for 10 minutes. Pellets were resuspended in 0.25 M sucrose supplemented with a protease inhibitor cocktail (Sigma Aldrich Inc.).
[Polyclonal Antibodies]
A rabbit anti-SOX6 affinity purified polyclonal antibody, which recognizes a peptide (amino acid residues 349 to 364) of human SOX6, was purchased from Chemicon International, Inc.
[Western Blot and Blocking Analysis for Confirming SOX6 Expression]
SOX6 expressions in glioma and normal brain were examined by the above-described assay. 20 μg of total protein was loaded into each lane. The first antibody was the rabbit antihuman SOX6 affinity purified polyclonal antibody (10 μg/ml), and the second antibody was the alkaline phosphatase-labeled goat anti-rabbit IgG (Fc) antibody (Cappel Inc.) diluted by 1:2000. For the blocking experiment, the diluted SOX6 polyclonal antibody (10 μg/ml) was incubated with 293T cells expressing SOX6 at a final concentration of 20 μg/ml in 1% PBS and 0.5% skim milk, and then subject to Western blotting as described above.
[Immunohistochemical Staining]
Tumor samples and normal brain tissue were fixed in 10% formalin and then embedded in paraffin for immunohistochemical analysis. Sections (5 μm) were cut and mounted onto microscope slides coated with poly-L-lysine. The mounted tissue sections were deparaffinized with xylene and rehydrated. Antigen revival was carried out as described in literature (J. Pathol. 183, 116-123, 1997). Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide in methanol, followed by washing in phosphate-buffered saline (PBS). The nonspecific binding of antibodies was blocked by incubation in 0.02 M PBS, containing 5% BSA, and PBS, containing 0.1% Triton X-100, for 1 hour. The slides were then incubated overnight at 4° C. with the antihuman SOX6 rabbit polyclonal antibody (1 μg/ml), which was diluted in the same blocking solution. The slides were incubated for 30 minutes at 37° C. with a second antibody (Universal Immuno-peroxidase Polymer, Anti-Rabbit antibody; Histofine Simple Strain MAX PO (R), made by Nichirei Corporation), and the HRP labeling was visualized using DAB. The sections were lightly counterstained with hematoxylin. Each step was followed with three washes in PBS.
For evaluation of the proliferative activity of the tumors, sections were stained with the monoclonal antibody, MIB-1. Dewaxed and rehydrated sections in a 10 mM citrate buffer of pH 7 were processed five times for 4 minutes each in a 600-watt microwave oven. After treatment for 1 hour at room temperature with 5% bovine serum albumin containing 3% horse serum, the sections were incubated overnight at 4° C. with MIB-1 (diluted by 1:50, made by Immunotech Inc.). Primary antibodies were visualized by the avidin-biotin complex technique using DAB. Sections were lightly counterstained with hematoxylin. Proliferating cell indexes were analyzed using a total of 1,000 or more tumor cells at three or more regions expressing the highest numbers of immuno-positive nuclei. The analysis was carried out using a computer-assisted image analyzer (Luzex F; Nireco Corp.).
[Analysis of Sera from Healthy Individuals, Glioma Patients, and Patients with Various Cancers for Examination of the Presence of SOX6-Specific IgG Antibodies]
Serum screening by SEREX was carried out on various patients to confirm the specificity and relevance of the antibody response to SOX6. Sera from 36 glioma patients, 14 patients of other brain disorders, 54 patients with various cancers, and 37 healthy individuals ranging from 20 to 78 years in age were diluted by 1:100 and used to screen IgG antibodies specific to SOX6. Transcripts of SOX6 induced serological responses in 12 (33.3%) of the 36 glioma patients. However, transcripts of SOX6 induced serological responses in 0 (0%) of the 14 patients of other brain disorders and only 1 (1.9%) of the 54 patients with other cancers. With sera obtained from 37 normal adults, no antibody against the abovementioned protein was detected with the exception of the serum from one individual (Table 4).
[The Immunogenic Epitope of the SOX6 Protein]
Since it has been suggested that the conserved HMG box domain, which is a DNA binding site of SOX6, may be recognized by IgG derived from glioma patient sera, the sequence encoding the HMG box of SOX6 was subcloned into the pET32a vector to prepare a His-tagged recombinant protein. First, the seroreactivity against the HMG box of SOX6 was examined by Western blot analysis. The IgG in sera obtained from three of eight glioma patients, who were found by SEREX to exhibit positive seroreactivity against SOX6, detected a 30 kDa protein corresponding to the HMG box expressed by recombination (
[Analysis of SOX6 Gene Expression in Glioma and Normal Tissues by RT-PCR and Northern Blotting]
In order to examine the SOX6 gene expression amounts in glioma and normal adult tissues, the differences of SOX6 gene expression between glioma and normal adult brains were evaluated by quantitative RT-PCR using SYBR Green. In comparison to a normal adult brain, SOX6 was expressed in significantly large amounts in all glioma tissues that were analyzed (
It has been reported that two transcripts, one long and one short, are detected from the mouse SOX6 gene (Proc. Natl. Acad. Sci. USA, 97, 4180-4185, 2000; Embo. J. 17, 5718-5733, 1998) and the human SOX6 gene (Gene 265, 157-164, 2001). The difference in the lengths of these transcripts is most likely caused by the different sizes of both the 3′ and 5′ untranslated regions (Embo. J. 17, 5718-5733, 1998). The short transcript is specifically expressed in adult testis, and the long transcript is expressed in mouse brain (Nucleic Acids Res. 23, 3365-3372, 1995) and chondrocytes (Embo. J. 17, 5718-5733, 1998) during development but disappear from these tissues in adults.
To investigate which transcript is expressed in glioma, the expression of the SOX6 mRNA in human glioma was determined by Northern blotting (
[Selective Expression of the SOX6 Protein in Glioma]
The expression of the SOX6 protein was evaluated by Western blotting using a polyclonal antibody. The antibody detected the 90 kDa band of the SOX6 protein in 293T cells transfected with the SOX6 cDNA. On the other hand, the antibody did not detect the same band in 293T cells that were not transfected by the SOX6 cDNA (left side of
The specificity of the abovementioned polyclonal antibody was confirmed by incubating the 293T cells that express SOX6 with the antibody. The 90 kDa signal in the immunoblot disappeared in this blocking experiment using the polyclonal antibody (right side of
[Immunohistochemical Analysis of SOX6 in Glioma]
Using the antibody against SOX6, sectioned materials from formalin-fixed tumors were analyzed, and the proliferative activity was evaluated using the MIB antigen. All of the analyzed eighteen types of glioma tissues expressed SOX6 in nuclear tumor cells. On the other hand, SOX6-positive cells were not detected from nonneoplastic tissue derived from the cerebral cortex (
The correlation of SOX6 expression with histological malignancy, proliferation, and seroreactivity in glioma are summarized in Table 5. SOX6 expression was confirmed in all tumor tissues from patients having IgG antibodies against SOX6. Furthermore, though the possibility of a relationship between the expression of SOX6 and proliferative activity in glioma was evaluated, a clear correlation was not seen between the two.
aThe seroreactivities of SOX6 as determined by SEREX are indicated as +: positive; −: negative.
bThe proliferating cell indexes were analyzed in a total of 1000 or more tumor cells in three or more regions expressing the highest numbers of MIB-1 positive nuclei.
cThe staining patterns are described as −: negative or faint staining; +: positive in less than 30% of tumor cells; ++: positive in 30% to less than 70% of tumor cells; +++: positive in 70% or more of tumor cells.
NA: not available;
M: male;
F: female.
The above Example clearly shows SOX6 to be a new glioma expression antigen that causes an immunological response in no less than 33% of glioma patients. The high level of immunological response to the SOX6 protein is considered to be due to high expression of the SOX6 gene. Furthermore, though the expression of SOX6 in normal tissue is limited, it is expressed primarily in glioma tissue. The above results show that SOX6 can be a useful target for the development of diagnostic means and treatments for glioma.
The present invention's testis-derived glioma antigens and/or glioma antigen genes are useful for the treatment and diagnosis of glioma and other cancers and are also useful for obtaining fundamental knowledge concerning gliomagenesis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001-367960 | Nov 2001 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP02/12502 | 11/29/2002 | WO |
