WO2007037538A9 - Application therapeutique ou diagnostique du gene spo11 - Google Patents

Application therapeutique ou diagnostique du gene spo11

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Publication number
WO2007037538A9
WO2007037538A9 PCT/JP2006/320017 JP2006320017W WO2007037538A9 WO 2007037538 A9 WO2007037538 A9 WO 2007037538A9 JP 2006320017 W JP2006320017 W JP 2006320017W WO 2007037538 A9 WO2007037538 A9 WO 2007037538A9
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WIPO (PCT)
Prior art keywords
cancer
gene
protein
antibody
seq
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PCT/JP2006/320017
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English (en)
Japanese (ja)
Other versions
WO2007037538A1 (fr
Inventor
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
Original Assignee
Link Genomics Inc
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Link Genomics Inc, Shinichirou Niwa, Yasutaka Makino, Tomoki Ikuta, Kazuya Arai, Takayuki Shindou, Hiromichi Ogura filed Critical Link Genomics Inc
Priority to JP2007537778A priority Critical patent/JPWO2007037538A1/ja
Publication of WO2007037538A1 publication Critical patent/WO2007037538A1/fr
Publication of WO2007037538A9 publication Critical patent/WO2007037538A9/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • the present invention relates to a S P O 1 1 gene that is a gene specifically amplified in cancer, its therapeutic or diagnostic use, and the like.
  • Cancers Malignant tumors (cancers) are characterized by lethality due to generalization through proliferation, invasion, and metastasis. Local therapies such as surgical resection or radiation therapy cannot adequately address metastatic recurrent cancer, and the development of systemic pharmacotherapy is expected to improve the outcome of cancer treatment in the future. Yes.
  • Chemotherapy which is the current center of cancer drug therapy, often uses cell killing agents that directly act on the DNA and / or RNA of cancer cells and cause the cells to die. It also acted on normal cells such as bone marrow cells, germ cells, hair matrix cells, and gastrointestinal epithelial cells, and had strong side effects.
  • EGFR epidermal growth factor receptor
  • tyrosine kinase inhibitor iletsa gene: gefitinib
  • Herceptin gene: trastuzumab
  • HER-2 human epidermal growth factor receptor 2
  • Japanese colon cancer tends to increase year by year, and the number of deaths is lung cancer and stomach It is in 3rd place. By age, 60s is the most common, followed by 50s and 70s.
  • the cause of the increase in colorectal cancer may be genetic factors or environmental factors, but it has been pointed out that Western diets, especially excessive consumption of animal fat, may be the cause.
  • Development of an effective molecular target drug for colorectal cancer is awaited.
  • about half of the tumor markers used for diagnosis (CEA, CA 19-9) are positive even in advanced colorectal cancer, with no organ specificity, and higher performance diagnostic agents. Development is desired. Disclosure of the invention
  • the present inventors have determined that the gene that is frequently amplified in cancer (especially colorectal cancer) is the SPO l 1 gene. I found it.
  • the present inventors have further found that cancer cell growth can be suppressed by inhibiting the expression of SPO11 protein in colorectal cancer cell lines and cervical cancer cell lines, and to complete the present invention. It came. That is, the present invention provides the following cancer therapeutic agents, screening methods for candidate substances having a cancer suppressive action, cancer diagnostic agents, cancer diagnostic kits, cancer diagnostic methods, and the like.
  • a cancer therapeutic agent comprising a substance that inhibits the expression of the SPO1 gene as an active ingredient.
  • the cancer therapeutic agent according to (1) above comprising a substance selected from the group consisting of: (3) A cancer therapeutic agent comprising an S P O 1 1 protein activity inhibitor as an active ingredient.
  • the cancer therapeutic agent according to (3) above comprising a substance selected from the group consisting of:
  • a screening method comprising a step of selecting a compound that reduces the expression level as compared with a case where a test compound is not contacted.
  • a method of screening for an activity inhibitor of SPO11 protein comprising: (a) contacting the SPO l 1 protein with a test compound;
  • a screening method comprising a step of selecting a compound that binds to the SPO1 1 protein.
  • a cancer therapeutic agent comprising the antibody according to (9) above.
  • a cancer diagnostic agent comprising the antibody according to (9) above.
  • a diagnostic agent for cancer comprising a base sequence that can be hypridated under stringent high-prediction conditions in the SPO11 gene or a part of the base sequence thereof.
  • kits for cancer diagnosis containing a polynucleotide comprising a base sequence that can be hyper-predated under stringent high-precipitation conditions.
  • the biological sample is whole blood, serum, or plasma, (1 9) Method described in the above.
  • the diagnostic method according to (2 2) comprising:
  • a method for treating cancer comprising a step of administering an expression inhibitor of SPO11 gene to a patient.
  • a method for treating cancer comprising a step of administering a S P O 11 protein activity inhibitor to a patient. .
  • (3 0) Use of a SP 0 11 protein activity inhibitor for the manufacture of a medicament for the treatment of cancer.
  • a cancer therapeutic agent containing an SPO 11 gene expression inhibitor as an active ingredient which is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 1.
  • a cancer therapeutic agent comprising a polynucleotide having a nucleotide sequence of 0.
  • the present invention provides novel drugs, kits and methods useful for the treatment and / or diagnosis of cancer (for example, colorectal cancer), and a screening method for candidate compounds having cancer suppressive activity.
  • cancer for example, colorectal cancer
  • screening method for candidate compounds having cancer suppressive activity for example, cancer suppressive activity.
  • Fig. 1 is a histogram showing the frequency of gene amplification in 20 samples from patients with colorectal cancer of the SP 0 11 gene.
  • Fig. 2 is an optical micrograph (phase contrast image) showing the results of RNAI analysis when s siRNA of the SPO1 1 gene was transferred to the colon cancer cell line RKO.
  • FIG. 3 is a graph showing the results of evaluating the R N Ai effect by measuring the number of viable cells when the SPO11 gene siRNA was transfected into the colorectal cancer cell line RKO.
  • Fig. 4 is a photograph showing the results of verifying the RNAi effect using an optical microscope when the SPO l 1 gene siRNA was transfected into a cell line CCD 18 Co derived from normal colon tissue. (Phase contrast image).
  • Fig. 5 is a graph showing the results of verifying the RNA i effect by measuring the number of viable cells when the SPO l 1 gene si RNA was transfected into the cell line CCD 18 Co derived from normal colon tissue. is there.
  • Fig. 6 is a photograph showing the results of Northern hybridization performed using various normal organ tissues.
  • FIG. 7 is an optical micrograph (fluorescence image) of a portion (6 cells) of cancer cells in a sample tissue derived from a colon cancer patient analyzed by the FISH method.
  • FIG. 8A and FIG. 8B are graphs showing the results of (A) serum derived from a colon cancer patient and (B) serum derived from a healthy subject analyzed by mass spectrometry, respectively.
  • 9A to 9C show the correspondence between the peak shown in FIG. 8 and amino acids (or amino acid sequences) determined by MS / MS analysis.
  • FIG. 10 is a graph showing the results of verifying the R N Ai effect by measuring the number of living cells when s i R N A of the SPO 11 gene was transfected into a cervical cancer cell line HeLa cell line.
  • Fig. 11 is an optical micrograph (differential interference image) showing the result of observing the behavior of the experiment of Fig. 10 in time series under a microscope and in detail.
  • the inventors of the present invention verified a gene amplified by the array CGH method using a sample derived from a colon cancer patient, and identified a gene amplification region specific to colorectal cancer. Of the regions where amplification occurs frequently in the specimen, the human SPO l 1 (SPO 1 1 meioticroteincova 1 ent 1 yboundto DSB—like (S. cerevisiae)) gene is frequently encountered in specimens from colon cancer patients. I found out.
  • SPO 1 1 SPOl 1 meiotic protein covalently bound to DSB-like (S. cere 'e)
  • DSBs DNA double-strand breaks
  • Meiosis is a special type of cell division that occurs in the process of germ cells becoming haploid gametes in living organisms.
  • meiosis homologous chromosomes pair after DNA replication, diploid through the first division, and haploid at the second division. Therefore, 4 gametes are generated from 1 germ cell.
  • homologous chromosome pairing is under precise control. Occurs and recombination is a characteristic of sexual reproduction.
  • the first half of the first division is due to chromosomal morphological changes, depending on the morphological changes of the leptoten (filament), zygoten (combined), pakiten (thick), diproten (double), diakine (moving) ).
  • SC Synaptonemal Complex
  • the DNAs that are paired with the SC are aligned in the middle, chromosome pairing begins in the zygotene phase, and the pakiten phase is the period during which the SC is formed.
  • SC has a protein structure called Central element (CE) in the center and lateral element (LE) on the side.
  • CE Central element
  • L lateral element
  • Scp3 (Dobson, M. J., et al. (1994) J. Cell Sci. 107, 2749-2760) is known as a component of LE.
  • LE is formed in the leptotene period, and it is paired with the Zygoten period (Zickler, D., & Kleckner, N. (1998) Annu. Rev. Genet. 32, 619-697). It is formed by bridging LEs with fi laments (TFs),. It is formed during the Pakiten period until the Diproten period (Schmekel, K., et al. (1998) Chromosome Res. 6, 155-159).
  • SP 0 11 is homologous to the subunit of Topoisomerase VI (T0P6A), which is Type II topoi somerase. It has been reported that site-directed mutagenesis prevents homologous recombination during meiosis if a conserved tyrosine residue is mutated (Y135F) and Type II topoisomerase activity is lost (Bergerat, A , et al. (1997) Nature 386, 414-417).
  • SPO 11 acts directly with DSBs during meiosis (Neale, MJ et al. (2005) Nature 436, 1053-1057).
  • DSBs formation has been considered an important step in the early stages of canceration of cells. Abnormal stimulation of cell growth promotion can lead to stress during DNA replication and, in some cases, replication errors and DSBs. They are subject to repair by DNA damage checkpoints and growth inhibition by apoptosis.
  • canceration is promoted when checkpoint ⁇ does not function, such as when P53 is a mutant.
  • the formation of DSBs causes the destabilization of chromosomes, become and contribute to this in the promotion of cancerous (Gorgoul is, VG, et, al. (2005) Nature 434, 907-913) at 0 cancer
  • the expression of SPO11 may play an important role in cancer. '
  • Camptothecin Topicoi somerase I inhibitor
  • Doxorubicin Topico i somerase H inhibitor
  • SPO 11 is classified as DNA Topoi some rase, which is the same as Topoi some rase I and ⁇ , but has a different conformation (Gadelle, D. et al. (2003), BioEssays 25, 232-242).
  • l Low molecular weight compounds that inhibit the activity of 1 are promising as new anticancer agents.
  • the present inventors have also confirmed that the growth of cancer cells can be suppressed by suppressing the expression of the SP 0 11 gene by RNAi (RNA interference). Therefore, cancer can be treated by suppressing the expression of the SPO1 1 gene. It is also possible to diagnose cancer by measuring the expression level of the SPO1 1 gene.
  • RNAi RNA interference
  • the present invention comprises (1) an SPO 11 gene expression inhibitor as an active ingredient And (2) a cancer therapeutic agent containing an SPO 1 1 protein activity inhibitor as an active ingredient.
  • SP 0 1 1 gene refers to 1 8 2 6 bases registered under A ccession No .: NM— 0 1 2 4 4 4 on an NC BI nucleotide base. (Bergerat, A., et al. (1997) Nature 386, 414-417), but is not limited thereto, for example, in the nucleotide sequence of the gene.
  • High stringency conditions that are stringent to the base sequence of the gene or its complementary sequence, such as a variant that is altered by having one or more base substitutions, deletions, additions, or insertions
  • the gene consisting of a polynucleotide having a base sequence that can be hybridized below is also included in the “SP011 gene” as used herein.
  • Hypridization is a known method or a method similar thereto, for example, 'Molecular' Cloning (M o 1 ecu 1 ar C 1 oning T hird E dition, J. S a mb rooketa 1., C old S pring H arbor L ab. Press. 2 0 0 1).
  • stringent conditions may be any of low stringency conditions, medium stringency conditions and high stringency conditions.
  • Low stringency conditions are, for example, conditions at 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 32.
  • the “medium stringent conditions” are, for example, the conditions of 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, ⁇ 0% formamide, and 42.
  • “High stringent conditions” are, for example, conditions of 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, it does not affect the stringency of the hybridization. There are multiple factors that affect the temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., and those skilled in the art can select similar factors to achieve the same stringency. It can be realized.
  • the base sequence of SEQ ID NO: 1 for example, 70% or more, when calculated using the default parameters of homology search software such as FA SSTA, BLAST, etc. 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more , 9 7% or more, 98% or more, 99% or more of the polynucleotide having identity.
  • inhibition of gene expression means any event in a series of events from gene to protein production (for example, transcription (production of mRNA), translation (production of protein)) By inhibiting the production of the protein encoded by the gene.
  • SP0 1 1 protein refers to NC BI Yuno, °, 3 9 6 amino acids registered in the database as A ccession No .: N P — 0 3 6 5 7 6
  • a human SPO 1 1 protein consisting of residues (SEQ ID NO: 2) and substantially the same activity as this protein (for example, one or more activities selected from Type II topoisomerase activity)
  • a mutant protein consisting of an amino acid sequence in which deletion, substitution, insertion, and / or addition of one or more amino acid residues has occurred in the sequence.
  • the amino acid mutation site and number in the above mutant protein are not particularly limited as long as the mutant protein retains substantially the same activity as the original protein, but the number of mutations is, for example, 1 to 50. 1 to 40 pieces, 1 to 30 pieces,! ⁇ 2 5! ⁇ 2 0 ,! ⁇ 1 5 pcs! ⁇ 10 pieces, :! ⁇ Nine, :! ⁇ 8, 1-7, 1-6 (1 ⁇ several) ⁇ 5 pcs! ⁇ Four One, three, one, two, one. In general, the smaller the number of mutations, the better.
  • such a mutant protein has an amino acid sequence of SEQ ID NO: 2 and about 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 9 2 % Or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of amino acid sequences having identity
  • a protein having substantially the same activity as the original protein In general, the larger the homology value, the better.
  • the above-mentioned S P O 1 1 protein includes a “partial peptide” of S P O 1 1 protein.
  • the partial peptide of the SPO 1 1 protein is a partial peptide consisting of a partial amino acid sequence of the amino acid sequence of the SPO 1 1 protein (SEQ ID NO: 2), preferably the aforementioned SPO 1 1 protein Any one having the same activity as that of the protein may be used.
  • SEQ ID NO: 2 at least 20, preferably at least 50, more preferably at least 70, more preferably at least 100, most preferably Examples include polypeptides having an amino acid sequence consisting of at least 200 amino acid residues.
  • these polypeptides contain an amino acid sequence corresponding to the portion involved in the activity of the S P .O iI protein.
  • the partial peptide used in the present invention is one or more of the above-mentioned polypeptides in the amino acid sequence (for example, about 1 to 20 pieces, more preferably about 1 to 10 pieces, still more preferably 1 to 5 amino acid residues) may be changed by deletion, addition, substitution, or insertion.
  • the SP011 protein used in the present invention can be prepared from cells or tissues expressing the protein. Further, these proteins can be synthesized by a known peptide synthesizer, or can be prepared by a recombinant method using an appropriate host cell selected from prokaryotic organisms or eukaryotic organisms.
  • the SPO 11 protein used in the present invention may be derived from any species, but is preferably derived from rabbit. “Substantially the same activity” indicates that these activities are qualitatively equivalent.
  • the activity (such as Type II topoisomerase activity) is the same (for example, about 0.01 to; 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times)
  • quantitative factors such as the degree of activity and the molecular weight of the protein may be different. These activities can be measured according to known methods described in literatures such as Bergerat, A. et al. (1994) J. Biol. Chem. 269, 27663-27669. It can be measured according to the screening method described later.
  • cancer therapeutic agent includes anticancer agents, cancer metastasis inhibitors, cancer cell apoptosis inducers, cancer cell proliferation inhibitors, cancer cell infiltration inhibitors, cancer preventive agents, and the like. Used in meaning.
  • cancer (or cancer) and “tumor” are used as terms having the same meaning.
  • a cancer therapeutic agent containing an inhibitor of 1 S PO l 1 gene expression in one embodiment, the present invention provides a cancer therapeutic agent containing an SPO 11 gene expression inhibitory substance as an active ingredient.
  • SPO 11 gene expression inhibitor is not limited as long as it inhibits the expression of SPO ll gene.
  • SPO ll gene For example, (i) SP 01 1 gene to SPO 11 mRNA And (ii) a substance that inhibits translation from SPOl 1 mRNA to SP011 protein.
  • Examples of substances that inhibit transcription from S P O 1 1 gene to S P O 1 1 mRNA include
  • Examples of substances that inhibit translation from S P O l l mR N A to S P O 1 1 protein include
  • RNA i action on S P O l l mRNA or a part thereof for example, siRNA
  • nucleic acid means RNA or DNA.
  • nucleic acid may contain not only purine and pyrimidine bases but also those having other heterocyclic bases that have been modified. These modifications are methylated purines and pyrimidines, acylated Purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or ethers, amines, etc. It may be converted to a functional group.
  • RNA i is a phenomenon in which the expression of a foreign gene and a target endogenous gene are both inhibited when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell.
  • Examples of RNA used here include double-stranded RNA that causes RNA interference with a length of 19 to 30 bases, such as ds RNA (doublestrand RNA), si RNA (small interfering RNA). Or sh RNA ( shorthairpin RNA).
  • RNA can be locally delivered to a desired site by a delivery system such as ribosome, and can be locally expressed using the above-mentioned vector that generates double-stranded RNA force.
  • ds RNA, si RNA or sh RNA double-stranded RNA
  • the methods for preparing and using such double-stranded RNA are known from many literatures (Special Table 2 0 2-5 1 6 0 6 2; US Permission No. 2 0 0 2 Z 0 8 6 3 5 6 A; Nature Genetics, 2 4 (2), F e., 1 80-1 8 3; Genesis, 2 6 (4), Ap ril, 24 0-2 44; Nature, S pe. 2 1, 40 7: 6 8 0 2, 3 1 9-2 0; Genes.
  • the length of the double-stranded RNA exhibiting the RNA i effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21 to 25 bases, most preferably Is 2 1 to 2 3 bases.
  • the following si R N A (used in Example 3) can be used.
  • the term “antisense nucleic acid” or “antisense polynucleotide” has a polynucleotide that is complementary to at least a part of a DNA region of interest, and the polynucleotide is a small part of the region.
  • the term refers to a nucleic acid that can hybridize with a part.
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include nucleic acid sulfur derivatives, thiophosphate derivatives, and polynucleotide amides that are resistant to degradation of oligonucleotides. However, it is not limited to them.
  • the antisense nucleic acid to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
  • the nucleic acid thus prepared can be transformed into a desired animal using a known method.
  • the sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene of the animal to be transformed or a part thereof, but is completely complementary as long as the gene expression can be effectively suppressed. It doesn't have to be.
  • a complementary antisense sequence in the untranslated region near the 5 ′ end of the mRNA of SPO 1 1 gene is effective in inhibiting translation of the gene.
  • a sequence complementary to the coding region or 3 ′ untranslated region can also be used.
  • Antisense nucleic acid effective for inhibiting gene translation is about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 9% or more of the transcript of the target gene. 5% or more complementarity. .
  • the length of the antisense nucleic acid is at least about 10 bases (for example, about 10 to 40), preferably about 15 More than base, more preferably about 100 bases or more, and still more preferably about 500 bases or more.
  • Antisense nucleic acids can be designed with reference to known literature (for example, Hirashima and Inoue, Shinsei Chemistry Laboratory 2 Nucleic acid IV gene replication and expression, Japan Biochemical Society, Tokyo Kagaku Dojin, 1 9 9 3, 3 1 9-3 4 7), J. Kawakamieta 1., P harm T ech Japan. V ol. 8, p. 2 4 7, 1 9 9 2; V o 1. 8, p. 3 9 5, 1 9 9 2; see S. T. Crookeetal., Ed., Antisense Research and Applications, CRCP ress, 1 9 93 etc.).
  • a nucleic acid having a liposomal activity that specifically cleaves the transcript of the SP 0 11 gene is used as an active ingredient.
  • ribozyme activity refers to a nucleic acid that specifically cleaves mRNA, which is a transcription product of a target gene. Some ribozymes have a size of more than 400 nucleotides, such as group I intron type and Ml RNA contained in RNase P, but they have an activity of about 40 nucleotides called hammerhead type or hairpin type. Some have domains (Protein Nucleic Acid Enzymes, 1990, 35, p. 2 1 9 1).
  • FEBSL ett 1 9 8 8, 2 2 8, p. 2 2 8
  • FEBSL ett 1 9 8 8, 2 3 9, p. 2 8 5
  • protein nucleic acid Enzymes 1 9 90, 3 5, p-2 1 9 1
  • the hairpin type ribosome for example, Nature, 1 986, 3 2 3, p. 349; Nuc 1 A cids Res, 1 9 9 1, 1 9, p. 6 7 5 1; You can refer to Hiroshi Kikuchi, Chemistry and Biology, 1 992, 30, p.
  • a compound other than a nucleic acid that inhibits the transcriptional activity of the SP 0 11 gene can be used as an active ingredient.
  • a compound is, for example, a compound that binds to a factor involved in the expression / transcription of the SPO11 gene.
  • Such a compound may be a natural product or a synthetic compound.
  • Such a compound can be obtained by the screening method described below. 1. Cancer treatments that contain inhibitors of protein activity
  • the present invention also provides a cancer therapeutic agent containing a substance that inhibits the activity of S P O l 1 protein.
  • S PO 1 1 protein activity inhibitor includes, for example, (a) an antibody that binds to the SPO 1 1 protein,
  • antibody means an antibody that reacts with the full length or fragment of a protein.
  • the form of the antibody of the present invention is not particularly limited, as long as it binds to the SPO11 protein of the present invention, in addition to the polyclonal antibody and the monoclonal antibody, a human antibody, a human form by gene recombination.
  • antibody fragments, antibody fragments and modified antibodies thereof are also included.
  • Antibodies that bind to the SP011 protein can be prepared by methods known to those skilled in the art. Details of the anti-S PO 11 antibody will be described later.
  • SP 0 1 1 protein mutant having a dominant negative property with respect to S PO 1 1 protein means that an endogenous wild-type SP 0 1 is expressed by expressing a gene encoding the same. 1 Refers to proteins that have the function of eliminating or reducing protein activity
  • a compound other than the antibody or the mutant that binds to the SPO1 1 protein can be used as an active ingredient.
  • a compound is, for example, a compound that binds to the SPO11 protein and inhibits its activity.
  • a compound may be a natural product or a synthetic compound.
  • Such a compound can be obtained by the screening method described below.
  • the above-mentioned substance that can inhibit the activity of the SP 0 11 protein of the present invention can be used as a cancer therapeutic agent. 2. Screening method for substances that inhibit the activity or expression of SPO l 1 protein
  • the present invention also provides a method for screening a candidate compound having a cancer suppressing action.
  • One preferred embodiment is a method using as an index the binding between the SPO1 1 protein and the test compound.
  • a compound that binds to the SPO11 protein is expected to have an effect of inhibiting the activity of the SPO11 protein.
  • the compound preferably binds to the active site of the SPO11 protein.
  • the S.P011 protein is contacted with a test compound.
  • the SPO 11 protein can be used, for example, in a purified form of the SPO ll protein, in a form expressed intracellularly or extracellularly, or on an affinity column. It can be in a combined form.
  • the test compound used in this method can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • the binding between the SP011 protein and the test compound is then detected.
  • test compound used for this method.
  • natural compounds, organic compounds, inorganic compounds, proteins, peptides, etc. as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, oceans
  • compound libraries include, but are not limited to, biological extracts and plant extracts.
  • the binding between the SPO 1 1 protein and the test compound can be detected by, for example, a label attached to the test compound bound to the SP 0 1 1 protein.
  • a change in the activity of the SPO 11 protein caused by the binding of the test compound to the SPO 11 protein expressed intracellularly or extracellularly can also be detected as an indicator.
  • the binding activity between the protein and the test compound can be measured by a known method (for example, Type II Measurement of topoisomerase activity (Bergerat, A. et al. (1994) J. Biol. Chem. 269, 27663-27669.)).
  • test compound that binds to the SPO1 1 protein and inhibits its activity is then selected.
  • the compound isolated by this method is expected to have a cancer suppressing action and is useful as a cancer therapeutic agent.
  • Another embodiment of the screening method of the present invention is a method using the expression of S P O l 1 gene as an index.
  • a test compound is brought into contact with cells expressing the SPO1 1 gene.
  • cells expressing the SPO1 1 gene examples include cells derived from pets, livestock, etc., including but not limited to humans, mice, cats, dogs, sushi, hidges, and birds.
  • Cells that express the SPOII gene include cells that express the endogenous SPO11 gene, or cells that have the exogenous SP011 gene introduced and the gene is expressed. Can do.
  • a cell in which an exogenous SPO11 gene is expressed can usually be prepared by introducing an expression vector into which the SPO11 gene has been inserted into a host cell.
  • the expression vector, Yuichi can be prepared by general genetic engineering techniques.
  • test compound used in this method is not particularly limited.
  • natural compounds, organic compounds, inorganic compounds, single compounds such as proteins and peptides, compound libraries, expression products of gene libraries, Cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, etc. are used.
  • Contact of a test compound to a cell expressing the SPO 11 gene is usually performed by adding the test compound to the culture medium of each cell expressing the S PO 11 gene.
  • the test compound is a protein or the like
  • “contact” can be performed by introducing a DNA vector expressing the protein into the cell.
  • the expression level of the SP011 gene is then measured.
  • gene expression includes both transcription and translation.
  • the gene expression level can be measured by methods known to those skilled in the art. For example, mRNA can be extracted from cells expressing the SPO 11 gene according to a conventional method, and the transcription level of the gene can be reduced by performing Northern hybridization or RT-PCR using this mRNA as a saddle. Measurements can be made.
  • the promoter region of the SPO 11 gene is isolated according to a conventional method, and a gene that can be detected downstream using a labeled gene (for example, luminescence, fluorescence, color development, etc.
  • the transcription level of the gene can also be measured by observing the activity of the marker gene.
  • the protein fraction is collected from cells expressing the SPO 11 gene, and the level of gene translation is measured by detecting the expression of each SPO 11 protein by electrophoresis such as SDS-PAGE. You can also.
  • the antibody used for detecting the S PO 11 protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.
  • a compound that decreases the expression level is then selected as compared with the case where the test compound is not contacted (control).
  • Compounds selected in this way become candidate compounds for cancer therapeutics.
  • the present invention also provides an anti-SPOll antibody, a cancer therapeutic agent containing this antibody, and the like.
  • the cancer therapeutic agent is Used for cancer targeted therapy or targeted drug delivery.
  • anti-SPO 11 antibody includes an antibody that specifically binds to S PO 11 protein (including fragments (partial peptides) or salts thereof).
  • the anti-SPO11 antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in view of ease of purification.
  • the term “antibody” here is used to include any antibody fragment or derivative.
  • Fab, Fab ′ 2 , CDR humanized antibody
  • multifunctional antibody multifunctional antibody
  • single chain antibody S c F v
  • the antibody of the present invention can be produced by a known method. Methods for producing such antibodies are well known in the art (see, for example, Har 1 ow E. & Lane D., Antibody, Old Spring Laboratory Pres (1 9 8, 8)). See).
  • the protein used as the sensitizing antigen is usually SPO 11 protein or a salt thereof.
  • the SPO 11 protein includes a partial peptide thereof, which is not limited to, for example, a fragment of the amino acid sequence of SEQ ID NO: 2, for example, 20 or more, 40
  • the above is a partial peptide having 60 or more, 80 or more, 100 or more consecutive amino acid sequence portions.
  • these fragments for example, amino (N) terminal fragment and carboxy (C) terminal fragment are used.
  • one or more (preferably about 1 to 10, more preferably several (1 to 6)) amino acid residues in the above amino acid sequence are deleted. , Substitutions, insertions and / or additions There may be.
  • SP ⁇ 1 1 protein or its partial peptide salts include, for example, salts with inorganic acids (eg, hydrochloric acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid). Salt is used.
  • the SP011 protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or human, particularly from human. The protein is preferred.
  • SP01 protein (In the present specification, in the description of antibodies, these are collectively referred to as “SP01 protein”) and administered as an antigen to mammals such as rabbits, mice, and rabbits.
  • Dosage of antigen per animal is 0.1 ⁇ : when adjuvant is not used! O O mg, and l to 100 zg when an adjuvant is used.
  • the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Immunization is mainly performed by injecting intravenously, subcutaneously or intraperitoneally.
  • the immunization interval is not particularly limited, and immunization is carried out 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably at intervals of 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later.
  • antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, and spleen cells or local lymph node cells are preferred.
  • myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice can be used.
  • the cell line used has drug selectivity and can survive in HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state.
  • HAT selection medium including hypoxanthine, aminopterin, and thymidine
  • myeloma cells include mouse myeloma cell lines such as X 6 3 A g. 8. 65 3, NSIZ 1-A g 4-1, NSOZ l, and rat myeloma cell lines such as YB 2/0.
  • Cell fusion consists of 1 X 10 6 to 1 X 10 7 ml of antibody-producing cells and 2 X 1 0 5 to 1 in animal cell culture media such as serum-free DMEM, RPMI — 1 6 40 2 X 1 0 6 cells ml of the myeloma cells were mixed (the antibody producing cells with myeloma cells and cell ratio of 2: 1 to 3: 1 is preferred), the fusion reaction in the presence cells fusion promoter .
  • the cell fusion promoter polyethylene dalycol having an average molecular weight of 1000 to 600,000 Dalton can be used.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (eg, electrical mouth population).
  • the cell suspension is appropriately diluted with, for example, RPMI-1 64, 0 medium containing urchin fetal serum, and then plated on a microtiter plate about 3 ⁇ 10 5 Zwe 1 1 Add selective medium to each well, and then change the selected medium appropriately.
  • RPMI-1 64 0 medium containing urchin fetal serum
  • Zwe 1 1 Add selective medium to each well, and then change the selected medium appropriately.
  • Hybridoma screening may be performed in accordance with a normal method, and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by an enzyme immunoassay, a radioimmunoassay, or the like. Cloning of fused cells is performed by limiting dilution. Finally, a hybridoma, a cell that produces a monoclonal antibody that reacts with the SPO 11 protein, was constructed. Stand up.
  • a normal cell culture method or ascites formation method can be employed.
  • the hive re dormer about 1 X 1 0 7 cells were administered intraperitoneally to the mammal of the same species as the animal from which the myeloma cells are derived, thereby High Priestess dormer. The large quantities grown.
  • antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out, ion exchange chromatography, gel filtration, affinity chromatography, etc. are appropriately selected or combined. Can be purified.
  • the antigen described above is administered to mammals such as rats, mice, and rabbits.
  • the dose of antigen per animal is from 0.1 to LOO mg when no adjuvant is used, and from 10 to 100; ug when adjuvant is used.
  • adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and immunization is carried out 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks.
  • ELISA enzyme immunoassay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • an antibody (column adsorbed fraction) that reacts with the SPO1 1 protein is collected by subjecting the polyclonal antibody in the antiserum to an affinity ram fixed with the SPO1l protein.
  • the reactivity of the polyclonal antibody in the antiserum to the SPO1 protein can be measured by the ELISA method or the like.
  • the F ab or F ab ' 2 fragment can be prepared by digestion with a conventional protease (eg, pepsin or papain).
  • a conventional protease eg, pepsin or papain.
  • Humanized antibodies are described, for example, by Riec hma nn et al. (Riechmann JM o 1 B iol. Oct 5; 2 0 3 (3): 8 2 5-8, 1 9 8 8), Jones et al. Nature 3 2 1: 5 2 2-5 2 5, 1 9 8 6) —can be prepared by one of the methods—
  • Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 1. 6, No. 10, 1 9 8 8”, Japanese Patent Publication No. 3-7 3 2 80, etc.
  • human antibodies such as “Nature Genetics, Vol., 15, p. 1 4 6-1 5 6, 1 9 9 7”, “Nature Genet”
  • the antibody that binds to the SPO1 1 protein of the present invention can be used for the purpose of, for example, inhibiting the growth or migration of cancer cells.
  • a human antibody is preferably a human antibody in order to reduce immunogenicity.
  • an antibody When used as a diagnostic agent, an antibody may be labeled with a labeling substance (eg, a radioisotope or fluorescent substance) for monitoring. If necessary, it can be labeled with radioactive substances, fluorescent compounds, etc. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Similarly, the antibody SP011 antibody can be labeled using a bioluminescent compound. The presence of a bioluminescent protein is measured by detecting the presence of fluorescence. Important bioluminescent compounds for this purpose are luciferin, luciferase and aequorin.
  • a labeling substance eg, a radioisotope or fluorescent substance
  • the antibody of the present invention can be used for specifically detecting Spo1 1 protein and the like present in a subject such as a body fluid or tissue.
  • production of antibody columns used to purify SPO 1 1 protein, detection of SP 0 1 1 protein in each fraction during purification, SPO 1 1 protein in test cells It can be used to analyze the behavior of 3.2 Complexes containing anti-S P O 1 1 antibody, etc.
  • the anti-SPO11 antibody used in the present invention is an agent that can be an agent having a neutral activity that attenuates the activity of the antigen in the therapeutic agent or diagnostic agent of the present invention. ⁇ If necessary, it can be used in combination with other drugs to produce a therapeutic effect. Therefore, in another embodiment, the present invention provides an anti-SPO 1 1 antibody and another drug for use in targeted therapy or targeted imaging of cancer (eg, colon cancer). Also provided are composites, compositions containing such composites, and the like. According to such an embodiment, the anti-SPO 11 antibody used in the present invention is used to express other drugs that exhibit therapeutic effects or labeling agents for diagnosis, etc. Can be delivered to the target site.
  • Examples of the “other drug” used in the present invention include a viral vector or a non-viral vector for introducing a gene into a target, such as a radioisotope, a therapeutic protein, or a small molecule drug. Indicated.
  • examples of the “radioisotope” include radioactive halogen elements such as fluorine 1 18, iodine 1 1 2 5 ( 1 2 5 I), and iodine 1 3 1.
  • radioactive halogen elements can also be widely used as radiotherapeutic agents or radiodiagnostic agents by labeling antibodies and peptides in the same manner as the above-mentioned radioactive metal elements.
  • odorization with 1 2 5 I or 1 3 1 I can be bound to an antibody or antibody fragment by a known method such as the chloramine T method.
  • technetium one 9 9 m for the diagnosis indium one 1 1 1 and gallium - 6 7 (6 7 G a ) such, As the therapeutic Lee Tsu thorium one 9 0 (9 ° Y), rhenium one 1 8 6 ( 1 8 6 R e) or rhenium— 1 8 8 ( 1 8 8 Re) can be used.
  • a metal chelator is usually used.
  • Known metal chelating agents include EDTA, DTPA, diaminodithio compound, cyclam, and DOTA. These chelating agents may be pre-bonded to the antibody and then labeled with a radioactive metal, or may be labeled with the antibody after forming a radioactive metal chelate.
  • site force-in that activates cells responsible for immunity is suitable.
  • human interleukin 2 human granulocyte-macrophage colony-stimulating factor And human macrophage colony stimulating factor, human interleukin 12 and the like.
  • toxins such as ricin and diphtheria toxin can be used to directly kill colon cancer cells.
  • a cDNA encoding the fusion protein is constructed by linking the cDNA encoding the therapeutic protein to the cDNA encoding the antibody or antibody fragment, and this DNA is either prokaryotic or eukaryotic.
  • a fusion antibody can be produced by inserting the expression vector into a biological expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • small molecule drug is used herein to mean “radioisotope” or “therapeutic tongue”. It is used to mean a diagnostic or therapeutic compound other than “protein”.
  • small molecule drugs include alkylating agents such as naylogen mustard and cyclofasphamide, antimetabolites such as 5-fluorouracil and mesotrexe, daunomycin, bleomycin, mitomycin C, Antitumor agents such as antibiotics such as daunorubicin and doxorubicin, plant alcohols such as vincristine, vinblastine, and vindesine, and hormones such as evening moxifen and dexamethasone (Clinical Oncology (Japan Clinical Oncology) 1 9 9 6 Cancer and Chemotherapy))), or Steroids such as Hyde mouth cortisone and Prednisone, Non-Steroidal agents such as aspirin and Indomethacin, Immune chimerale, Penicillamine and other immunomodulators
  • daunomycin and antibody can be bound by binding between daunomycin and the amino group of the antibody via dartal aldehyde, or by binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carpositimide. And the like.
  • a viral vector modified so as to be capable of binding to the anti-SPO11 antibody of the present invention can be used (for example, an adenovirus vector (Wang, P., eta 1 (1 9 9 5) Somatic Celland Molec. Genet. 2 1, 4 2 9-44 1), retroviral vector (N aviaux RK, eta 1. (1 9 9 6) J. V irol 7 0, 5 7 0 1-5 7 0 5), lentiviral vector (N a I dini, L. (1 9 9 8) C urr. O pin. B iotechno 1. 9, 4 5 7-46 3))).
  • an adenovirus vector Wang, P., eta 1 (1 9 9 5) Somatic Celland Molec. Genet. 2 1, 4 2 9-44 1
  • retroviral vector N aviaux RK, eta 1. (1 9 9 6) J. V irol 7 0, 5 7 0 1-5 7 0 5
  • lentiviral vector N
  • virus vectors include cell proliferation-related genes, apoptosis-related genes, immune regulatory genes, and other target sites (eg, colon cancer), for example, cancer cells
  • a gene (therapeutic gene) that has a therapeutic effect such as inducing apoptosis is incorporated.
  • the virus vector that binds to the anti-SP011 antibody when administered to a patient in need of gene therapy with the anti-SP011 antibody, recognizes the antigen recognized by the anti-SPO11 antibody (ie, SPO11). Can be targeted to existing sites.
  • the anti-SPO1 1 antibody and the other drug can be combined chemically or genetically.
  • “chemical bond” includes ionic bond, hydrogen bond, covalent bond, bond by intermolecular force, bond by hydrophobic interaction, etc.
  • “genetic engineering bond” For example, the binding mode between an antibody and a therapeutic protein when a fusion protein consisting of an antibody and a therapeutic protein is produced using any technique such as genetic recombination is included.
  • a cancer therapeutic agent containing the S P O 1 1 gene expression inhibitor of the present invention a cancer therapeutic agent containing a S P O l 1 protein activity inhibitor,
  • PO l 1 antibody is either a viral or non-viral vector carrying a radioisotope, therapeutic protein, small molecule drug, and therapeutic gene, or any combination of these and chemical or genetic engineering Therapeutically bound therapeutic agents can be formulated based on known techniques.
  • a pharmaceutically acceptable carrier can be added as necessary according to a conventional method.
  • surfactants for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, taste masking
  • the present invention is not limited to these, and other commonly used carriers can be appropriately used.
  • Examples of the dosage form of the therapeutic agent of the present invention include tablets, powders, pills, powders, granules, fine granules, soft and hard capsules, film coating agents, and pellets as oral agents.
  • Agents, sublingual agents, pastes, etc., parenteral agents include injections, suppositories, transdermal agents, ointments, plasters, liquids for external use, etc.
  • the optimum dosage form can be selected.
  • An inhibitor of the activity of the S PO 11 protein (or the expression of the S PO 11 gene) as an active ingredient can be contained in the preparation in an amount of 0.1 to 99.9% by weight.
  • the dose of the active ingredient of the drug of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally for patients (as 60 kg) From about 0.1 mg / day to: I, 0,000 mg, preferably from about 1.0 to: 100 mg, more preferably from about 1.0 to 50 mg.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • patient for 60 kg About 0.1 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 O mg per day is conveniently administered by intravenous injection. .
  • the final decision can be made as appropriate based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
  • the preparation thus obtained can be administered, for example, to humans and other mammals (eg rat, rabbit, hidge, buyu, ushi, cat, inu, monkey, etc.). it can. In the case of animals other than humans, the amount converted per 60 kg can be administered.
  • the therapeutic agent of the present invention is cancer (for example, colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder).
  • Prevention, treatment of uterine cancer eg cervical cancer, endometrial cancer
  • testicular cancer thyroid cancer
  • knee cancer ovarian cancer
  • brain tumor blood tumor, etc.
  • the agent of the present invention contains an SP 0 1 1 protein activity inhibitor or SPO 1 1 gene expression inhibitor as an active ingredient, it is used as an anticancer agent, cancer metastasis inhibitor, cancer cell apoptosis inducer, etc. Can do.
  • the target cell, tissue, organ, or cancer type is not limited to a specific type.
  • the agent of the present invention is both an SPO 1 1 protein activity inhibitor and an SP 0 1 1 gene expression inhibitor. May be included.
  • the antisense nucleic acid when used, the antisense nucleic acid is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus virus vector, etc. Thereafter, it can be administered according to known means.
  • Antisense nucleic acids can be formulated alone or with a physiologically acceptable carrier and administered via a catheter such as a gene gun or a hydrogel catheter.
  • a combination of a virus vector such as a recombinant adenovirus particle and an anti-SPO11 antibody when used for cancer treatment, these may be used alone, but in general, Used with a pharmaceutically acceptable carrier.
  • a carrier a carrier as described above and an aqueous isotonic solution such as water, physiological saline, glucose, and human albumin are preferable.
  • additives, preservatives, preservatives, balance, etc. that are usually used in pharmaceutics can also be added.
  • the pharmaceutical composition thus prepared can be administered by an appropriate administration form and administration route depending on the disease to be treated.
  • administration forms include emulsions, syrups, force capsules, tablets, granules, injections, ointments and the like.
  • Treatment of the anti-SP 0 1 1 antibody-one virus vector particle of the present invention or a pharmaceutical composition comprising the same In general, it is preferable to administer 10 3 to 10 15 virus particles at a time per adult, but this may vary depending on the disease state and the nature of the target cell / organization. Good.
  • the number of administrations may be from once to several times a day, the administration period may be from one day to several months or more, and one set is set as one to several injections, and many sets are administered intermittently over a long period of time. May be.
  • virus vector single particle or virus vector single nucleic acid molecule used in the present invention can be used for detection of specific cells and / or tissues, or diagnosis of disease states.
  • a viral vector obtained by incorporating a detectable marker gene into a nucleic acid molecule of a viral vector and transfecting it into an appropriate host cell.
  • a detectable marker gene obtained by incorporating a detectable marker gene into a nucleic acid molecule of a viral vector and transfecting it into an appropriate host cell.
  • it can be used for detecting and diagnosing tumor cells by binding a detectable label to the anti-SP011 antibody. 5.
  • the present invention also provides a diagnostic agent for cancer.
  • the diagnostic agent for cancer of the present invention comprises (a) an antibody against SPO 11 protein, or (b) a stringent sequence in the SPO 11 gene or a part of its nucleotide sequence. It contains a polynucleotide consisting of a base sequence that can be hyperpredable under high predation conditions.
  • the diagnostic method using the anti-SPO11 antibody of the present invention includes, for example, (a) a step of bringing a subject-derived biological sample into contact with an antibody against the SPOll protein, and (b) Detecting and Z or quantifying the binding of the antibody to the SPO11 protein or a partial peptide thereof or a salt thereof in the sample.
  • the labeled anti-SPO 1 1 antibody is used to detect and z or quantitate the binding between the SPO 11 protein or a fragment thereof and the anti-SP 0 11 antibody.
  • subject-derived biological sample refers to a subject-derived tissue, cell, or body fluid (for example, blood (including whole blood, plasma, serum, etc.), urine, lymph, saliva, sweat, semen, etc. ) including.
  • a “subject” is usually a human subject who is or is expected to undergo a cancer screening, such as a human subject who has or is suspected of having cancer. included. Examples of such cancers are colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer. , Uterine cancer (eg, cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, spleen cancer, ovarian cancer, brain tumor, blood tumor, etc. Is preferred.
  • Immunoassays to detect the expression of SPO 11 in biological samples derived from subjects as described above are collected from subjects who are suspected of having cancer (eg, colorectal cancer) and at risk for cancer. And subjecting the biological sample to contact with an anti-SPO11 antibody under conditions that produce specific antigen-antibody binding, and then measuring the amount of immunospecific binding by the antibody. Such antibody binding is used to detect the presence and / or increased expression of the SP 0 11 protein. In this case, detection of increased SPO1 1 protein expression is an indicator of disease state. If necessary, the level of S P O l 1 protein in the biological sample may be compared to the level of healthy individuals who do not have cancer.
  • a biological sample such as a serum sample is contacted with a solid support or carrier such as nitrocellulose for the purpose of immobilizing all proteins present in the sample.
  • the support is then washed with buffer and subsequently treated with detectably labeled anti-SP011 antibody.
  • the solid support is then washed twice with buffer to remove unbound antibody.
  • the amount of bound antibody on the solid support is determined according to well-known methods taking measurement. The detection conditions suitable for each measurement can be appropriately determined by those skilled in the art using conventional test methods.
  • the antibody is conjugated to an enzyme, such as that used in the enzyme immunoassay (EIA) [Voi 1 er, A By “Enzyme-linked immunosorbent assay” (ELISA), 1 9 7 8, Diagnostic Horizons, 2: 1-7, Microbiological A ssociates Qu arterly Pub 1 ication, Wa lkersvi 1 le. M'D; V oi 1 er, A. by J. C li n. Pathol., 3 1: 5 0 7-5 2 0, 1 9 7 8: B utier, J. E., Me th h.
  • EIA enzyme immunoassay
  • Enzymes that bind to antibodies can be visualized, for example, by spectrophotometry.
  • an appropriate substrate preferably a chromogen.
  • Enzymes that can be used to label the antibody with a detectable label include, but are not limited to, peroxidase and alkaline phosphatase. This can be achieved by a colorimetric method using a chromogenic substrate for the enzyme.
  • RIA radioimmunoassay
  • sandwich immunoassay immunometric method
  • FIA fluorescence immunoassay
  • TRFIA time-resolved fluorescence immunoassay
  • EIA enzyme Immunoassay
  • LIA Luminescent immunoassay
  • ELIA Electrochemiluminescence immunoassay
  • Latex agglutination Immunoprecipitation Atssey, Precipitation reaction, Gel diffusion precipitation reaction, Immunodiffusion assay
  • Examples include an agglutination assay, a complement binding assay, an immunoradiometric assay, a fluorescence immunoassay, and an immunoassay selected from the group consisting of a protein A immunoassay (WO 0 1) No. 4 2 27 No. 3 page 9 9 line 25 to page 4 2 line 8 EP 1 1 1 1 1 04 7 A No. 2 paragraph [0 1 1 5] page 19 Lines 35 to 20 (see page
  • diagnosis of various diseases associated with dysfunction of S PO 11 protein can be performed by utilizing the in vivo SP 0 1 protein quantification method using the antibody of the present invention.
  • Can do For example, if an increase in the concentration of SP protein is detected, it may be caused by, for example, a disease caused by overexpression of SPO ll protein (eg, cancer (eg, colorectal cancer)). It can be diagnosed as high or likely to be affected in the future.
  • the anti-SP011 antibody of the present invention can also be used for diagnosis by i n v i v o.
  • the preparation and use of antibody preparations that can be used here are well known in the art. For example, an antibody-chelate glaze agent is described in Nuc 1. Med. Biol.
  • a probe or primer designed based on the base sequence of the SPO11 gene can be used.
  • a diagnostic method can, for example, (a) be capable of hyperpredation under high-precipitation conditions stringent to a biological sample derived from a subject and the base sequence of the SPO 11 gene or a fragment thereof.
  • a step of quantifying is a step of quantifying.
  • the DNA of the SPO 11 gene (or gene fragment thereof) in a biological sample derived from a subject is detected using the probe. And / or quantify.
  • the length of the base sequence used as a probe is, for example, 12 bases or more, 15 bases or more, 18 bases or more, 21 bases or more, 24 bases or more, 27 bases or more, 30 bases or more, or even longer It may be a polynucleotide fragment of length.
  • the low, medium or high stringency conditions described above may be used.
  • a base sequence (antisense polynucleotide) complementary to the base sequence of the SPO11 gene or a fragment thereof is also included.
  • Methods of hybridization of probes and nucleic acids are known to those skilled in the art, such as International Publication No. 8 9 0 6 6 9 8, EP—A 0 2 0 0 3 6 2, US Patent No. 2, 9 1 5, 0 8 2, EP_A 0 0 6 3 8 7 9, EP-A 0 1 7 3 2 5 1, EP—A 0 1 2 8 0 1 8
  • a target sequence can be detected or quantified using a known method using a specific polynucleotide probe or primer for the SP 0 11 gene.
  • known methods include Southern hybridization, Northern hybridization, RT-PCR method, PCR-SSCP method (Genomics, Vol. 5, 8 7 4 Pp. 8 7 9 (1 9 8 9)), Proceedingsofthe National Academy of Sciences of United States of America, Vol. 8 6, 2 7 6 6-2 7 7 0 (1 9 8 9 years))), FISH method, DNA chip or array GH (Com parative Genomic Hybridization) method can be used. Quantitative detection can be performed by quantitative RT-PCR.
  • the array C GH method is an application of the chromosomal C GH method (K allioni em i, A. eta 1. (1 9 9 2) Science 2 5 8, 8 1 8-8 2 1). DN with high density spotted genomic DNA fragments (BAC, PAC, YAC, etc.) covering the chromosomal region
  • BAC high density spotted genomic DNA fragments
  • YAC YAC
  • This is a method to detect abnormal DNA copy number in cancer with high resolution (Pinke 1, D. eta 1. (1 9 9 8) N at. G enet.
  • the mRNA level of the SPO11 of the cell is measured using a standard gene (housekeeping gene (eg, Shaper, N. L. et al., J. Mammary G land B iol. Neoplasia 3 (1 9 9 8) 3 1 5-3 2 4; Wu, Y. Y. and Rees, J. L.. It can also be compared with the mRNA level of rm. V enereo 1.80 (2 0 0 0) 2—3), preferably by RT-PCR.
  • a standard gene housekeeping gene (eg, Shaper, N. L. et al., J. Mammary G land B iol. Neoplasia 3 (1 9 9 8) 3 1 5-3 2 4; Wu, Y. Y. and Rees, J. L.. It can also be compared with the mRNA level of rm. V enereo 1.80 (2 0 0 0) 2—3), preferably by RT-PCR.
  • the target sequence (DNA, mRNA, etc.) is detected and quantified by the above-mentioned method and overexpression of the SP ⁇ 1 1 gene is confirmed, for example, a disease caused by overexpression of SPO1 1 (for example, it can be diagnosed that the cancer is likely to be cancer (eg, colorectal cancer)) or is likely to be affected in the future.
  • a disease caused by overexpression of SPO1 1 for example, it can be diagnosed that the cancer is likely to be cancer (eg, colorectal cancer)) or is likely to be affected in the future.
  • the presence of a target protein or a fragment thereof in a test sample can be identified using a mass spectrometer (MS). That is, by using a mass spectrometer, it is possible to determine the amisoacid sequence of the target protein or a fragment thereof, and determine whether or not the SPO 11 protein is present in the biological sample derived from the subject. be able to.
  • mass spectrometry a sample such as protein or peptide is ionized using MS, separated according to the obtained mass Z charge (m / z), and the intensity of the sample is measured to determine the mass of the sample. It is. So From the results of mass spectrometry, individual amino acids constituting the amino acid sequence of proteins and peptides can be identified.
  • ionization various methods such as matrix assisted laser desorption (MAL DI), electrospray ionization (ESI), gas phase (EI, CI), field desorption (FD), etc.
  • MAL DI matrix assisted laser desorption
  • ESI electrospray ionization
  • EI gas phase
  • FD field desorption
  • an ion separation method compatible with the ionization method is used.
  • a time-of-flight type timeoff 1 ight: TO F
  • ESI a quadrupole Mass spectrometers such as type (QMS), ion trap type, and magnetic field type are used.
  • Mass spectrometers are sometimes used in tandem.
  • sequencing using a sequencer eg, gas phase sequencer
  • sequencing using a sequencer eg, gas phase sequencer
  • the present invention also provides a kit for detecting and / or quantifying the SP0 ⁇ 1 protein or a fragment thereof in a body fluid sample of a subject containing an anti-SPO11 antibody as a cancer marker.
  • the SPOll gene or a fragment thereof in a biological sample derived from a subject which contains a base sequence that can be highly prehybridized under stringent high prehybridization conditions in the SPOll gene or a part of the base sequence.
  • kits for detecting and / or quantifying as a cancer marker are used to detect the best of cancer by the above-described immunological technique or the hyperpredation method.
  • cancers examples include colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, and uterus.
  • cervical cancer, endometrial cancer testicular cancer, thyroid cancer, spleen cancer, ovarian cancer, brain tumor, blood tumor, etc.
  • colorectal cancer is particularly preferable.
  • cancer marker refers to a subject's body fluid (eg, blood, urine, lymph, saliva, sweat, semen, etc.) or cells or tissues that are not derived from normal tissues, or This refers to a molecule that is selectively upregulated in cancer cells or tissues, and the presence of the molecule in the body fluid or cells or tissues of a subject indicates or suggests the presence of cancer.
  • the kit of the first embodiment contains a component for detecting and / or quantifying SPO1 1 antigen (including SPO1 1 protein and its partial peptide) in a body fluid sample from a subject.
  • a component for detecting and / or quantifying SPO1 1 antigen including SPO1 1 protein and its partial peptide
  • SPO 1 1 protein is detected and Z or quantified by ELISA
  • such a component can detect and / or detect the level of SP 0 1 1 in, for example, a tissue section or a body fluid sample such as blood or urine.
  • Such antibodies may be labeled with radioactivity, fluorescence, colorimetry, or enzyme labeling.
  • the kit of the present invention may contain a labeled secondary antibody.
  • the kit according to the second embodiment contains a polynucleotide having a base sequence that can be hybridized under high hybridization conditions stringent to the SPO11 gene or a partial base sequence thereof.
  • the kit of the present invention may contain the above-mentioned polynucleotide immobilized on a DNA chip.
  • the kit of the present invention comprises, in addition to a base sequence that can be highly pre-polymerized under anti-SPO11 antibody, SP011 gene or a part of its base sequence under high-precipitation conditions, a container and It may contain a label.
  • the label on or with the container may indicate that the drug is used to detect colorectal cancer markers.
  • other items such as instructions for use may be further included.
  • Example 1 Identification of colon cancer specific amplification gene by array C GH method
  • sample preparation of 200 colorectal cancer specimens and verification based on the array CGH method were performed.
  • NC BI Genetic Information
  • SPACC 11 SPO ll me iotic proteincovalently bound to DSB— 1 ike (S. Cerevisiae)
  • the gene was found to be frequently and highly elevated in colorectal cancer patients ( Figure 1 and Table 2).
  • Example 2 Verification of gene amplification in a colon-derived cultured cell line
  • the amplification in a colon cancer-derived cultured cell line was verified for a high-frequency gene region in colon cancer patients. did.
  • the cultured cell lines used were Caco 2 and RKO, which are cell lines derived from colorectal cancer.
  • Genomic DNA was extracted from the cultured cells according to the protocol attached to the kit using BLOOD & CELCUlTureDNAKit (QIAGEN).
  • Table 3 shows the degree of amplification (GZ R value) of the S P O l 1 gene in a cell line derived from colorectal cancer. As shown, in the colon cancer-derived cell line, it was found that amplification occurred in the SP 0 1 1 gene located in B AC Clone RP 1 1 — 6 71 P 16.
  • Quantitative PCR was performed to confirm the amplification of the SPO11 gene region. Quantitative PCR can be performed using S YB RG reen RT-PCRR eagents (A pp 1 ied B iosys te rn s) according to the attached protocol. A pp 1 ied B iosyst ern s) It was. The following sequences were synthesized (outsourced to OPER ON) and used as primers.
  • Table 4 shows the degree of amplification of the SPO1 gene in a colon cancer-derived cell line. Values are relative to control DNA (normal). As shown in the figure, it was found that also in the colon cancer-derived cell line, amplification occurred in the SPO11 gene region.
  • the SPO l 1 gene which was frequently amplified in 200 colorectal cancer patients, was a colon cancer-derived cell line, and gene amplification was observed at the genomic level.
  • RNA i analysis was performed and the phenotype was observed.
  • the cell line was purchased from ATCC and cultured according to the attached protocol.
  • s i RN A a specific 21 mer within the gene was selected and s 1 RNA was synthesized with the sequence as the target (commissioned to QIAGEN).
  • the guide of siRNA into RK0 cells was Lipofectamine (Invitrogen), and 5 OnM siRNA was introduced into the cells according to the attached protocol call.
  • 5 OnM siRNA was introduced into the cells according to the attached protocol call.
  • N egative Control 1 siRNA QI AGE N was used. The cells were observed under an inverted microscope for 4 days after introduction into the cells.
  • siRNA is verified at the mRNA level using the quantitative RT-PCR method. From the cells 24 hours after introduction of siRNA, total RNA is extracted according to the attached protocol using Micro-t o -Mid iT o ltR N APu r if i c i t i o n S y s t ern (I nv i t ro g e n).
  • Quantitative PCR is performed using SYBRG reen RT—PCRR eagents Use (A pplied B iosyste rn s) and follow the attached protocol 7 7 500 0 Real -Time PCRS ystem
  • G lyceraldehyde — 3 phosphatedehydrogenase (GA PDH) Control Reagents (A pplied Biosyste rn s) as the standard gene for calculating the relative ratio, and calculate the relative ratio of GA PDH. . Measurement of viable cell count>
  • RNAi analysis of the SPO11 gene was performed using RKO, a cell line derived from colorectal cancer.
  • Fig. 2 shows the observed images on day 4 after Transfection of the SPOll gene siRNA in RKO cells (upper: X40; lower: X2200).
  • a, b, and c are 3 types of siRNA of the SPO 11 gene, respectively, and NC indicates a negative control.
  • NC N egative Control
  • Fig. 3 shows the results of measuring the number of viable cells using the measurement reagent on the 4th day after Transfection of SPO 11 gene siRNA to RK0 cells. The graph shows the relative amount to NC.
  • N egative Control 1 NC
  • Example 4 Functional analysis by RN Ai analysis using normal colon-derived cell lines
  • RN A 1 analysis was performed using cell lines derived from normal tissue of the large intestine, thereby suppressing the target gene. Has been verified to be cancer specific.
  • CCD18Co purchased from ATC was used. The culture conditions were in accordance with the attached protocol. The c sequence of Example 3 was used as the s 1 RNA used.
  • Lipofect iNe2200 Invitrogen was used, and 25 nM of siRNA was introduced into the cells according to the attached protocol.
  • N ega tiv e C o n t r o l s i RNA Q I AG EN was used. The cells were observed under an inverted microscope for 5 days after introduction.
  • RNA 1 in the SP ⁇ 1 1 gene a cell line CCD 1 8 Co derived from normal colon tissue, are as follows.
  • FIG. 4 shows an observation image obtained on the 5th day after siRNA of the SPO11 gene was transformed into CCD18Co cells (upper: X40; lower: X2200).
  • S P O l l c is a s i R N A 1 species of the S P O l l gene (c sequence of Example 3), and N C represents a negative control.
  • phenotypic observations were similar to NC and no effect was observed (Figure 4).
  • FIG. 5 shows the results of measuring the number of viable cells with the measurement reagent using the cells on the 5th day after the S rRNA of the SPO11 gene was transformed into CCD18Co cells by Transfection.
  • the graph shows the relative amount with respect to NC.
  • NC N ega tiv corn ol
  • the organ-specific expression of the SP011 gene in normal tissues was evaluated by the Northern hyperprecipitation method known in the art.
  • RNA Probe Using MTN Multiple Tissue Northern Blots (Clontech), hybridization was performed with 1,190 nt (NM_012444: 94–1284) RNA Probe according to the attached protocol.
  • RNA level expression in normal organ tissues 23 organs (Heart, Brain, Placenta, Lung) (Lung), Liver, Skeletal Muscle, Kidney, Pancreas, Spleen, Thymus, Prostate, Testis, Ovary (Ovary), Small Intestine, Colon, Peripheral Blood Leukocyte, Stomach, Thyroid, Spinal Cord, Lymph Node, Trachea ( Trachea), adrenal gland, and bone marrow were analyzed by the Northern hybridization method. As described above, the SPO ll gene sequence is registered in NCBI (http://www.ncbi.nlni.nih.gov) as Accession No.
  • Fig. 7 shows optical micrographs (fluorescence images) of some of the cancer cells (for 6 cells) observed in each specimen tissue (J). As shown, more than 3 spots were found in cancer cells. It was confirmed that the SPO11 gene region was amplified in 10 specimens whose gene amplification degree (G / R) was 1.2 or more by the array CGH method. It was also shown that gene amplification occurred from the early stage to the late stage of the disease state. This indicates that the SPOll gene region can be applied not only as a molecular target for cancer therapeutics but also in cancer diagnosis by FISH.
  • Example 7 Detection of SP 0 11 protein in blood by mass spectrometry
  • Serum specimen of colorectal cancer patient 10L and healthy subject serum specimen lO ⁇ L are diluted with 500L of diluted buffer solution (10mM Tris HC1 h 7.4 + 150mM NaCl), then ProteomeLab IgY-12 SC proteome partitioning kit (BECKMAN COULTER: A24618) was used to remove albumin, globulin, and other blood and serum proteins. Dithiothreitol (Wako: 049-08972) was added to the obtained fraction to a final concentration of 10 mM, and the reduction reaction was performed at 60 for 30 minutes.
  • diluted buffer solution 10mM Tris HC1 h 7.4 + 150mM NaCl
  • ProteomeLab IgY-12 SC proteome partitioning kit (BECKMAN COULTER: A24618) was used to remove albumin, globulin, and other blood and serum proteins. Dithiothreitol (Wako: 049-08972) was added to the obtained fraction to a final concentration of 10
  • Fig. 8A and Fig. 8B show the results of analysis of (A) serum derived from colorectal cancer patients and (B) serum derived from healthy subjects by the methods described above.
  • FIGS. 9A to C show the correspondence between the peak shown in FIG. 8 and amino acids (or amino acid sequences) determined by MS / MS analysis.
  • an ion peak corresponding to the fragment ion of the target molecule was clearly observed in the serum from colorectal cancer patients. That is, as shown in FIG. 9B, a partial sequence called IT FA PAE was determined at least from the C-terminal side, which was determined from positions 6 6 to 7 2 in the amino acid sequence (SEQ ID NO: 2) of the S PO ll protein. It corresponds to the amino acid sequence of the position. Therefore, as shown in FIG. 9A, a target peptide fragment (SEQ ID NO: 13) having amino acid sequences 66 to 75 was identified.
  • SPO 11 gene knockdown showed a growth-inhibiting effect, but the effect of cervical cancer cell lines was evaluated using the RNAi analysis method described above. .
  • RNAi analysis was performed using the above three siRNAs.
  • O 1 igofe tame ine (Invitrogen) was used, and 100 nM of siRNA was introduced into the cells according to the attached protocol.
  • Nega tiv corn ol s si RNA (Q I AGE N) was used. Clue 3 ⁇ 4
  • Fig. 10 shows the results of measuring the number of viable cells (MTT assay) using the reagent on the 4th day after Transfection of SPO11 gene siRNA to HeLa cells.
  • the graph shows the relative amount with respect to NC (Negative control siRNA (Qiagen)).
  • NC Negative control siRNA (Qiagen)
  • the present invention provides cancer therapeutic agents, diagnostic agents, diagnostic methods, therapeutic methods, kits used therefor, and the like. Therefore, the present invention is useful in fields such as cancer diagnosis or targeted therapy.

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Abstract

L'invention concerne un agent thérapeutique du cancer contenant un inhibiteur d'expression ou un inhibiteur d'activité de la protéine SPO11. L'invention concerne également un procédé de criblage d'un composé pouvant servir d'ingrédient actif d'un tel agent thérapeutique; un anticorps contre la protéine SPO11; un agent diagnostique du cancer et un procédé de diagnostic du cancer utilisant ledit anticorps et analogue.
PCT/JP2006/320017 2005-09-30 2006-09-29 Application therapeutique ou diagnostique du gene spo11 WO2007037538A1 (fr)

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