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WO2007037538A1 - Application therapeutique ou diagnostique du gene spo11 - Google Patents

Application therapeutique ou diagnostique du gene spo11 Download PDF

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Publication number
WO2007037538A1
WO2007037538A1 PCT/JP2006/320017 JP2006320017W WO2007037538A1 WO 2007037538 A1 WO2007037538 A1 WO 2007037538A1 JP 2006320017 W JP2006320017 W JP 2006320017W WO 2007037538 A1 WO2007037538 A1 WO 2007037538A1
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Prior art keywords
cancer
gene
protein
antibody
therapeutic agent
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English (en)
Japanese (ja)
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WO2007037538A9 (fr
Inventor
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
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Link Genomics Inc
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Link Genomics Inc
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Priority to JP2007537778A priority Critical patent/JPWO2007037538A1/ja
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Publication of WO2007037538A9 publication Critical patent/WO2007037538A9/fr
Anticipated expiration legal-status Critical
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    • 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 gene that is specifically amplified in cancer, its therapeutic or diagnostic use, and the like. Background art
  • Malignant tumors are characterized by lethality due to proliferation, invasion, and metastasis. Surgical excision or radiation Local therapy is sufficient for the treatment of metastatic recurrent cancer, and the development of drug therapy is expected to improve future cancer treatment results.
  • Some chemotherapy often acts directly and / or on RNA, killing cells to death, but other than cancer cells, such as bone marrow cells, living cells, gastrointestinal epithelial cells It had strong side effects on the cells.
  • recent molecular cell organisms, mechanisms related to cancer cell invasion, proliferation, metastasis, etc. are attracting attention as molecular targets that specifically act on the specific mechanism of cancer cells.
  • Representative examples include treatment of non-small cell lung cancer with GFR (epidermal growth factor receptor), tyrosine kinase inhibitor sa (generic name. Gefitinib) (W 9 6 3 3 9 8 0), It is in 3rd place.
  • GFR epidermal growth factor receptor
  • tyrosine kinase inhibitor sa generic name. Gefitinib
  • the cause of the increase in colorectal cancer and environmental factors may be considered, but it has been pointed out that this is due to the westernization of eating habits and excessive consumption.
  • the large intestine is waiting for the development of targeted drugs.
  • CEA CA 19-9 used for diagnosis only indicates that it is advanced colorectal cancer, has no organ specificity, and is a higher performance diagnostic agent. Disclosure of the invention
  • the present inventors have found that the P O 1 1 gene is frequently amplified in intensive studies (particularly colorectal cancer).
  • the present inventors have completed the present invention capable of suppressing the growth of cancer cells by inhibiting SPO1 1 in cell lines and cervical cancer cell lines. That is, the present invention provides a therapeutic agent, a screening agent for a candidate substance having a cancer suppressing action, a kit for cancer diagnosis, a method for diagnosing cancer, and the like.
  • a substance that inhibits the activity of SP 0 11 protein comprising a substance selected from the group consisting of:
  • a screening method comprising selecting a compound that binds to the SPO11 protein.
  • a cancer therapeutic agent comprising the antibody according to (9) above.
  • a cancer diagnostic agent comprising the antibody according to (9) above.
  • a cancer diagnostic agent which can be hyper-predated under a high pre-hydration condition in the SPO 1 1 gene or a part of the base sequence thereof.
  • SPO 1 1 gene or a part of its base sequence can be highly prehydidized under high hybridization conditions The method described in 9).
  • the cancer is colorectal cancer, (2 5)
  • (2 7) A method for treating cancer, comprising administering an SPO 11 gene expression inhibitor to a patient.
  • (3 1) A polynucleotide having the base sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10
  • SP 0 1 1 gene expression inhibitor is an active ingredient and therapeutic agent, and has a base sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 9, or SEQ ID NO: 10 A cancer therapeutic agent containing.
  • the present invention provides novel drugs, kits and methods useful for the treatment and treatment of cancer (eg, colorectal cancer), and methods for screening candidate compounds for cancer.
  • cancer eg, colorectal cancer
  • Fig. 1 is a histogram showing the frequency of the SP 0 1 1 gene relative to the degree of amplification of the 2 0 0 gene derived from colorectal cancer patients.
  • Figure 2 shows the results (phase contrast image) of the RNAI analysis when the SP 0 11 gene was transferred to the colorectal cancer cell line RKO.
  • Fig. 3 is a graph showing the results of RNA i obtained by measuring the number of viable cells in the colon cancer cell line RKO when s transfer of the SP 0 11 gene was performed.
  • Fig. 4 is a photograph (phase contrast image) showing the results verified by RNAi microscopy when the S child s1 RNA was transfected into the cell line CCD18Co derived from normal colon tissue.
  • FIG. 8A and FIG. 8B are graphs connecting the serum derived from (A) and the serum derived from (B) healthy subjects analyzed by mass spectrometry.
  • FIG. 9 A- shows the correspondence between amino acids (or amino acid sequences) determined by MS / MS analysis
  • Fig. 10 shows SP 0 1 1 NA in cervical cancer cell line HeLa cell line
  • FIG. 6 is a graph showing the results of further verifying the RN Ai effect when transfecting.
  • FIG. 11 is an optical micrograph showing the results of detailed observation of the experiment of FIG. 10 in time series under a microscope (the best mode for carrying out the differential interference invention).
  • the number of genes amplified in colorectal cancer was verified using specimens from cancer patients, and 1 (SPO ll me iotic proteinco 1 yboundto DSB — Like (S ciae)) The gene was found to be high in specimens from patients with colorectal cancer.
  • SPO 1 1 SP011 me lot ic protein shared ly boun (S cerevjsjae)
  • S cerevjsjae S cerevjsjae
  • the first half of the first division is divided into five stages, depending on the morphological changes of the chromosome: leptozygoten (fitting), pakiten (thick), and diplodiakinesis (migratory). Homology results in a structure called Synaptonemal Complex (SC) (1996) Curr Opin Cell Biol 8, 389-396).
  • SC Synaptonemal Complex
  • S C has a protein structure called Central element (CE) and its side element (LE) in the center.
  • CE Central element
  • LE is formed in the leptene phase and is zygo (Zickler, D, & Kleckner, N (1998) Annu Rev Ge 697).
  • CE is formed by cross-linking between LE by Transverse f containing Scpl from the Zygoten period, and is formed until the Diproten period (Schmekel, K, et al (1998) Chro 155-159) o
  • SPO l 1 is homologous to the subunit of Top (T0P6A), which is a Type II topoi somerase.
  • T0P6A is a Type II topoi somerase.
  • Site-directe reports that a mutation in a conserved tyrosine residue (Y135F) eliminates the topoisomerase activity, resulting in a homologous combination during meiosis (Bergerat, A, et al 386, 414). -417) 0
  • SPO l 1 is testis-specific in normal human tissues (Romanienko, PJ & Camerini-Otero, RD (1999) 156-169, Shannon, M, et al (1999) FEBS Lett 462, testis-specific expression. Molecules that have been recognized in recent years have been called Cancer-Te (CTA) and are candidates for cancer-specific antigens (Scanlan JM ⁇ Zo (2004) Cancer Immunity 4, 1 M & Erenschena, J (2005) Cancer Cell Internatio 11) Traditionally used as a serum antigen for colorectal cancer (2002) Cancer Res 62, 6750-6755)
  • the expression of SP011 in 0 cancer may have a role in cancer.
  • Topoisomerase I inhibitor Topical lsomerase I inhibitor
  • Doxorubicin , ⁇ lsomeras increase cancer cells by inhibiting ⁇ poisomerase activity.
  • S ⁇ 1 1 is classified as the same DNA Top as Topoisomerase I and ⁇ . Since the three-dimensional structure is different (Gadelle, De BioEssays 25, 232-242), compounds that inhibit the activity of SP 0 1 1 are promising as new anticancer agents.
  • the present inventors were able to suppress the growth of cancer cells by suppressing the expression of the SP 0 11 gene by RNA i (R. Therefore, the present inventors treat the suppression of the expression of the SP 0 1 gene.
  • SP 0 1 1 genetics And (2) SP 0 1 1 TAN A cancer therapeutic agent containing a harmful substance as an active ingredient.
  • SP 0 1 1 gene refers to a human SP (SEQ ID NO: 1) consisting of 1 8 2 6 bases registered under Accession No. ) (Bergerat, A, et al (1997) 414-417), but is not limited to this, for example, it is altered by substitution, deletion, addition, or insertion of one or more bases in the gene.
  • the base sequence of SEQ ID NO: 1 when compared with the base sequence of SEQ ID NO: 1, when calculated using a homology search software such as FAST, 75% or more, 80% or more, 8 5% or more, 90% or more 92% or more, 93% or more, 94% or more, 95% or more, 97% or more, 98% or more, 99% or more I can give you.
  • a homology search software such as FAST, 75% or more, 80% or more, 8 5% or more, 90% or more 92% or more, 93% or more, 94% or more, 95% or more, 97% or more, 98% or more, 99% or more I can give you.
  • inhibition of gene expression refers to inhibiting any one of a series of events from gene to protein (for example, including transcription (generation of mRNA) and quality). The production of the protein encoded by this gene should be inhibited.
  • SP 0 11 protein refers to a protein consisting of 3 96 amino acid residues registered under Accession No 6 5 7 6 (SEQ ID NO: 2) and the substance (eg, selected from Type II topoisomerase activity), and retains 1 amino acid residue deletion, substitution, insertion, and Z or addition to the amino acid sequence of this protein. Arise One, three, one, two, one. The number of mutations is generally good. In addition, such a mutant protein has about 70% or more, 75% or more, 80% or more, 85% or more 91% or more, 92% or more, 93% or more, 94% %, 96% or more, 97% or more, 98% or more, 99% or more of the same amino acid sequence and substantially the same protein as the original protein.
  • SP 0 1 1 protein includes SP 0 1 protein tide.
  • SP ⁇ 1 1 protein partial peptide P0 1 1 is a partial peptide consisting of a single amino acid sequence of the amino acid sequence (SEQ ID NO: 2), and preferably has the same activity as the activity of the 1 1 protein. That's fine.
  • SEQ ID NO: 2 amino acid sequence
  • at least 20 amino acids represented by SEQ ID NO: 2 preferably at least 50, more than 70, more preferably at least 100, at least 20 An amino acid sequence consisting of individual amino acid residues such as peptide.
  • amino acid corresponding to the part involved in the activity of these polypeptides Preferably, the amino acid corresponding to the part involved in the activity of these polypeptides.
  • the partial peptide used in the present invention has a deletion of one or more (for example, about 1 to 10, more preferably about 1 to 10 and even more preferable) amino acid residues in the amino acid sequence described above. , Addition, substitution, or insertion. “Substantially the same activity” means that the activity is characteristic. Therefore, it is preferable that the activity (Type II topoisomerase and the like (eg, about 0 to 1: L 0 0 times, preferably about 0 and more preferably about 0 to 5 times). The measurement of these activities can be performed in accordance with known methods described in the literature such as Bergerat, A et al (1994) J 269, 27663-27669. For example, screening methods described later Can be.
  • the identity of the amino acid sequence and nucleotide sequence is determined by the algorithm based on the Carl B LA ST (proc N at 1 S cl USA 8 7 2 2 6 4-2 2 6 8, 1 9 9 0; at 1 A cad S ci USA 9 0 5 8 7 3, based on B LA ST algorithm
  • a program called N or BLAS TX has been developed hu 1 SF, eta 1 .JM ol B iol 0 3,1 9 9 0).
  • cancer therapeutic agent refers to an anti-cancer agent
  • the present invention provides a cancer therapeutic agent containing SPO 11 gene as an active ingredient.
  • examples of substances that harm SP0 1 1 protein from S PO l l mRNA include:
  • a polynucleotide having SPO ll mRNA or a part thereof, or It may contain substituted purines and pyrimidines, acylated purines, or other heterocycles.
  • Leosides and modified nucleotides may also be sugar moieties, for example, one or more hydroxyl groups may be halogenated, substituted, or functionalized such as ether, amine.
  • RNA i is a phenomenon in which the expression of a foreign endogenous gene is inhibited when a double-stranded RNA that is identical or similar to the target gene sequence is introduced into the cell. , 19 to 30 bases long Double-stranded RNA, such as ds RNA (doub 1 ed RNA) sl RNA (sma 1 1 interfe NA) or sh RNA (shorthairpin R.
  • ds RNA doub 1 ed RNA
  • sl RNA sma 1 1 interfe NA
  • sh RNA shorthairpin R.
  • RNA is Delivery cis It is also possible to deliver locally to the desired site, and to express it locally using the vector as generated above
  • double-stranded RNA ds RNA, sl RNA or preparation method, use The method is well known from many literatures 2-5 1 6 0 6 2, USS Open Permit 2 0 0 2 Z 0 8 6 ature Genetics, 2 4 (2), Fe 0-1 8 3 ; G enesis, 2 6 (4), Ap r SAA pr 3 0, 9 9. 9 6 04 7-6 0 5 re B lotechnoogy V o 2 0 y, 4 9 7-5 0 0 Nature B iotech V o 2 0 5) ay, 5 0 0-5 0 8
  • the double-stranded RN having the RNA 1 effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably ⁇ 25 bases, and most preferably 21 to 23 bases.
  • s 1 RNA used in Example 3 was used (Table 1).
  • antisense nucleic acid or “antireotide” refers to a nucleic acid having at least a polynucleotide of a DNA region of interest and capable of hybridizing with a part of the polynucleotide.
  • Antisense nucleic acids can be RNA, DNA, or modified. However, it is not limited to them.
  • the antisense nucleic acid to be used is a suitable promoter, and preferably the nucleic acid prepared as described above containing a sequence containing a transcription termination signal on the 3 'side can be transformed into this using a known method.
  • the sequence of the antisense nucleic acid is not a sequence complementary to the endogenous gene of the trait or a part thereof, but may be omitted as long as the gene expression can be effectively suppressed.
  • a complementary antisense sequence near the 5 'end of the mRNA of the SP011 gene is designed, gene translation is blocked. It can be complementary to the coding region or the 3 'untranslated region. About 70% or more, preferably about 8 more preferably about 90% or more, and most preferably about 95% or more, with respect to the transcript of the antisen gene effective in inhibiting gene translation.
  • the length of the antisense nucleic acid is at least about 10 bases (about 40), preferably about 15 bases or more, and more than 100 bases or more. Yes, more preferably about 500 bases or less of this antisense nucleic acid can be designed by referring to known literature. Hirashima and Inoue, Shinsei Chemistry Laboratory 2 Nucleic acid IV gene Japan Biochemical Society, Tokyo Chemical Dojin, 1 9 9 3, p 3 1 9 K aw ak am ieta 1, P harm T ech Can.
  • ribozyme activity refers to site-specific cleavage of mRNA, which is a transcription product of a gene.
  • Some ribozymes have a 40-degree active domain called hammerhead type or hairpin type, such as group I intron type or M RNA, which has a capacity of more than 400 nucleotides (protein nucleic acid 3 5 P 2 1 9 1).
  • FEBSL ett, 1 9 8 8, 2 2 8, p 2 2 L ett, 1 9 8 8, 2 3 9, p 2 8 5, protein 9 0, 3 5, ⁇ 2 1 9 1; Nuc 1 A cids R 1 7, p 7 0 5 9 etc. can be referred.
  • N ature 1 9 8 6, 34 9, N uc 1 A cids R es, 1 9 9 1, 1 9 1; Hiroshi Kikuchi, Chemistry and Biology, 1 9 9 2, 3 0 , P 1 1 2.
  • the gene can be inherited by specifically cleaving a transcript of one gene in the present invention using such a ribozyme.
  • a compound that inhibits the transcription activity of the SP 0 11 gene can be used as an active ingredient.
  • it is a factor involved in the expression and transcription of the SP 0 11 gene.
  • Such compounds can be natural products or synthetic compounds, and can be obtained by the screening method described below. (a) an antibody that binds to SP 0 1 1 protein,
  • antibody means the full length protein or antibody.
  • the form of the antibody of the present invention includes not only the above polyclonal antibody, but also a human antibody, a recombinant body, an antibody fragment thereof, and an antibody modification product, as long as it specifically binds to SPO11 protein.
  • Antibodies that bind to SP can be prepared publicly by those skilled in the art. The anti-SP011 antibody will be described later. -
  • SP 0 1 protein variant having a property of being dominant to SP 0 1 protein means that the activity of endogenous wild-type S protein is lost by expressing the gene Alternatively, it has a function to reduce
  • SP ⁇ 1 Inhibits protein activity or expression Leaning method
  • the present invention also provides a screen for a candidate compound having a cancer suppressing action.
  • SPO11 protein has an effect of inhibiting the activity of SPO1 protein.
  • the compound is an SPO11 protein.
  • S PO 1 is brought into contact with a test compound.
  • the S PO I l protein can be, for example, a purified form of S PO 1, a form expressed intracellularly or extracellularly, or a form bound to an affinity column, depending on the indicator for detecting the binding of This compound can be appropriately labeled as necessary. Examples thereof include radiolabels and fluorescent labels.
  • test compound used for this method.
  • the compound isolated by this method has 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 S P O l 1 as an index.
  • a compound that expresses the SP011 gene is contacted.
  • cells examples include, but are not limited to, pet, and house cells such as Su, Cat, Inu, Hushi, Hedge, Tri.
  • an endogenous SP011 gene cell, or an exogenous SP011 gene introduced into the cell can be used.
  • Exogenous SP 0 1 1 cells can usually be prepared by introducing the inserted SP 0 1 1 gene into the host cell.
  • Test compounds used in this method are not particularly limited, but compounds, organic compounds, inorganic compounds, proteins, pep compounds, and compound libraries, gene libraries, cell extracts, cell culture supernatants, fermentation microorganism products, Marine raw extracts are used. To do.
  • the expression levels of transcriptional and translational genes can be measured by methods known to those skilled in the art.
  • the level can be measured by extracting from a cell that expresses the SP 0 11 gene according to the Northern blot method or RT-PCR method using this mRNA as a cage.
  • the SPO l motor region can be isolated according to a conventional method, and downstream thereof, there can be mentioned genes that can be detected using the luminescence of labeled residues luciferase, GFP, galactosidase, etc.
  • the bell can also be measured by looking at.
  • the compound selected as a compound that decreases the expression level as compared with a roll that is not contacted with a test compound is then used for a cancer therapeutic agent.
  • anti-SP011 antibody includes a specific substance in a fragment (partial peptide) of SP011 or a salt thereof.
  • the anti-SP011 antibody used in the present invention may be an monoclonal antibody or a monoclonal antibody, and the class of the body is not particularly limited, such as IgG, IgM, Ig or IgE.
  • the antibody having any isotype is IgG or IgM, and is preferably IgG for ease of purification.
  • antibody used herein is meant to include any antibody fragment or derivative, and includes, for example, Fab ′ 2 , CDR, humanized antibody, multifunctional antibody, single-chain antibody, and the like.
  • the antibody production method of the present invention is known in the art (eg, E & Lane D, Antibody, Coing Harbor Laboratory Pre 8). )
  • the present invention is a protein P11 protein used as a sensitizing antigen or a salt thereof.
  • the above SP 0 11 includes its partial peptide, which is limited to a fragment of the amino acid sequence of SEQ ID NO: 2, for example There may be.
  • the salt of SP 0 1 protein peptide used here include inorganic acids (eg, hydrochloric acid or organic acids (eg, acetic acid, formic acid, propionic acid), etc. Used as a sensitizing antigen for antibody acquisition.
  • the protein of the present invention is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammalian mouse or human, particularly preferably from human.
  • the immunization interval is not particularly limited, and it is immunized 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably weekly. Is collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later.
  • antibody-producing cells examples include spleen cells and lymph node cells, but spleen cells or local lymph node cells may be used. Furthermore, it is preferable that it can survive only in a state fused with antibody-producing cells.
  • myeloma cells include rat myeloma cell lines such as mouse mye YB 2 Z 0 such as X 6 3 ANSI / 1-A g 4-1, NS 0Z 1, and then the above myeloma cells and antibody-producing cells.
  • Cell fusion is performed in serum-free DMEM, RPMI — 1 6 40 cell culture medium, 1 X 10 6 to 1 X 10 7 pieces of Zm 1 and 2 X 1 0 5 to 2 X 1 0 6
  • the cell ratio between the production cell and the myeloma cell of 2.1 ml of myeloma cells and the myeloma cell is fused in the presence of a cell fusion promoter.
  • Cell fusion-promoting molecular weight polyethylene glycol having a molecular weight of 100 to 60,000 daltons can be used.
  • antibody-producing cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electon).
  • a normal cell culture method or ascites formation method can be used.
  • 10 containing hypridoma R PM I — 16 40 medium, MEM medium or serum-free medium In normal culture conditions (for example, 37 ° (: 5% 7-1) Cultivate for 4 days, and obtain antibody from the culture supernatant in the case of administering about 1 X 10 7 lydoma of mammals and allogeneic animals derived from myeloma cells, Collect ascites after 2 weeks Collecting the above-mentioned antibody If purification of the antibody is required, select an ammonium sulfate salting-out method, ionic luffy, gel filtration, affinity chromatography, or a combination thereof. You can do it.
  • the above antigen is administered to mammals such as rats and mice.
  • the dose of the antigen per animal is from 0 1 to: LOO mg when using an adjuvant, and to 100 g using an adjuvant.
  • adjuvants include Freuvant (FCA), Freund's incomplete adjuvant (FI aluminum adjuvant, etc.
  • Immunization is performed mainly by injecting subcutaneously or intraperitoneally. Blood is collected on the day when the antibody titer is shown to obtain an anti-clot.
  • the polyclonal antibody in the antiserum is applied to an affinity column fixed with a protein, and an antibody (column adsorption fraction) that reacts with SP is collected.
  • the reactivity of the polyclonal antibody in the antiserum against S P can be measured by, for example.
  • the Fab or Fab 2 fragment can be prepared by digestion with pepsin or papain (for example, using conventional methods).
  • Humanized antibodies are described, for example, by R 1 ec hma nn et al. (R in JM o 1 Biol Oct 5, 2 0 3 (3) 1 9 8 8), and J ones et al. (J ones et al. Nat 1.5. 2 2-5 2 5, 1 9 8 6).
  • Chimeric antibodies are, for example, “Experimental Medicine (Extraordinary Supplement 16, No 10, 1 9 8 8”), Japanese Patent Publication No. 3-7 3 2 8 80 humanized antibodies, for example, “Nature Genetic” 1 5, p 1 4 6— 1 5 6, 1 9 9 7 ”,“ Naturelcs, Vol 7, p 1 3— 2 1, 1 9 94 ”, Japanese Patent No. 3 65, International Publication W ⁇ 9 4-2 5 5 8 No. 5 public, June, 40th to 50th pages, 1 995 years, Vol 3 6 8, p 8 5 6— 8 5 9, 1 9 9 4 ”and Japanese Patent Publication No. 2 3 3 etc., respectively. Label with radioactive material or fluorescent compound as necessary.
  • a bioluminescent compound can be used as well as an antibody SPO 11 antibody, as well as fluoresceinate, rhodamine, phycoerythrin and fluore.
  • the presence of a bioluminescent protein is measured by the presence of fluorescence.
  • Bioluminescent luciferin, luciferase and aequorin are important for this labeling purpose.
  • the antibody of the present invention is used for specifically detecting 11 protein or the like in a sample such as a body fluid or tissue, or used for purifying SP 0 11 protein or the like. It can be used to detect SPO 1 1 protein, etc. in each fraction of time, and to analyze the behavior of SP 0 1 1 protein. 3 2 Complex containing anti-SP0 1 1 antibody, etc.
  • the anti-SP011 antibody used in the present invention may be an agent that itself has a weakening activity against an antigen in a diagnostic agent, or other agent for exerting a necessary effect.
  • the present invention in another aspect, provides a complex of PO 1 1 antibody and another drug used for targeted therapy or targeted imaging of cancer (example). Also provides such composites. According to such an aspect, the present invention Indicated.
  • examples of the “radioisotope” include iodine- 1 2 5 ( 1 25 1) and iodine 1 1 3 1 elements. These radioactive halogen elements can also be widely used as radiotherapeutic diagnostic agents by labeling antibodies and peptides in the same manner as the above elements.
  • the 1 25 I or 1 de reduction by known methods of chloramine-T method can be antibodies or binding.
  • indium 1 1 1 1 and gallium 6 7 6 7 Ga for treatment, yttrium 9 0 ( 9 0 Y), rhenium 6 R e) Or, when rhenium- 1 8 8 C 1 8 8 R e) or the like is used to label an antibody with a radioactive isotope, is usually used.
  • metal chelating agents EDTA, D-Nodizio compound, cyclam, and DOTA are known to be bound in advance to the antibody and then released, and after forming a radioactive metal chelate, it binds to the antibody. There is a law.
  • site force-in that activates is suitable.
  • human 2 human granulocyte-macrophage colony-stimulating factor, and colony-stimulating factor Human interleukin 1 2 etc.
  • ricin and diph toxins can be used to directly kill colon cancer cells.
  • therapeutic protein It may mean a diagnostic or therapeutic compound other than “protein”.
  • small molecule drugs include alkylating agents such as nitrogen cyclophosphamide, antimetabolites such as 5-flumesotrexe, daunomycin, mitomycin C, daunorubicin, doxorubicin-like vincristine, Hormone such as vinblastine, vindesine, hormonal agents such as moxifen, dexamethasone Bed tumor oncology (Edited by Japan Clinical Oncology Society 1 9 9 6 Years Cancer or steroids such as cortisone, prednisone, indometa Anti-inflammatory agents such as non-sterolide agents such as syn, immunomodulators such as gold thiamalamine, immunosuppressive agents such as cyclophosphamide, and chlorophenamine, maleic acid chlorate and anti-histamines Yakuhin Publishing Co., Ltd.)
  • Daunoma examples of the binding method include a method of binding between the amino group of the antibody and the antibody via dartal aldehyde, the amino group of dauno
  • virus vector 1 a virus vector modified so as to be capable of binding to anti-SP of the present invention can be used.
  • Denovirus vector Wang, P, eta 5) Somatic Celland Molec 2 1, 4 2 9-44 1
  • retrovirus vector Incorporates genes that have therapeutic effects such as inducing apoptosis.
  • Virus that binds to anti-SP ⁇ 1 1 antibody Injected to patients who need gene therapy together with SP ⁇ 1 1 antibody Antigen recognized by anti-SP ⁇ 1 1 antibody (ie, can be targeted to SP011 site) .
  • the anti-SP011 antibody and the other drug can be chemically or conjugated.
  • “chemical bond” is assumed to be due to ionic covalent bond, bond due to intermolecular force, and hydrophobic interaction, and “genetic engineering bond” is referred to as a fusion protein composed of, for example, a protein.
  • the binding mode between the antibody and the therapeutic protein should be used when the product is produced by genetic recombination.
  • Cancer treatment containing a PO 1 1 protein activity inhibitor containing the SP 0 1 gene expression inhibitor of the present invention A therapeutic agent containing an SPO 1 1 antibody, or the PO 1 1 antibody according to the present invention is radioactive.
  • Therapeutic agents that are genetically engineered with isotopes, therapeutic proteins, viral vectors or non-carriers carrying low and therapeutic genes, or any combination of these should be known techniques Can do.
  • Examples include cyclopropyl cellulose, hydroxypropyl methyl cell, disulfide lugetilamino acetate, polypinyl latin, medium-chain fatty acid triglyceride, polyoxyethylene oil 60, sucrose, carboxymethyl cellulose, corns, etc. .
  • Examples of the dosage form of the therapeutic agent of the present invention include oral powders, pills, powders, granules, fine granules, soft * hard capsules, pellets, sublinguals, and pastes.
  • non-propellants, suppositories, transdermal agents, ointments, plasters, and liquids for external use have optimal dosage forms according to the route of administration and subjects of administration.
  • Inhibitors of SP P 1 1 1 protein activity (gene expression) inhibitors as active ingredients can be from 0 1 to 9 9 9 in the drug product.
  • the dose of the active ingredient of the drug of the present invention varies depending on the administration subject, subject administration method, etc.
  • about 0 l mg to l per day for general (as 60 kg)
  • the single dose varies depending on organs, symptoms, administration method, etc. For example, is usually about 30 mg per day for patients (for 60 kg)
  • the therapeutic agent of the present invention is cancer (for example, colorectal cancer, stomach cancer, cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen bladder cancer, uterine cancer (eg cervical cancer) Uterine body cancer), adenocarcinoma, knee cancer, ovarian cancer, brain tumor, blood tumor, etc.)
  • cancer for example, colorectal cancer, stomach cancer, cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen bladder cancer, uterine cancer (eg cervical cancer) Uterine body cancer), adenocarcinoma, knee cancer, ovarian cancer, brain tumor, blood tumor, etc.
  • it is used for the prevention / treatment of colorectal cancer.
  • the agent of the present invention contains an SP 1 1 protein activity inhibitor 1 1 gene expression inhibitor as an active ingredient, a cell, tissue, organ used as a cancer metastasis inhibitor, cancer cell apoptosis inducer, etc.
  • the agent of the present invention may contain both the SP 0 1 protein activity inhibitor PO 1 1 gene expression inhibitor.
  • antisense Use nucleic acid Sense nucleic acid alone or after being inserted into a retrovirus vector, actor, adenovirus associated virus vector, and administered according to known means They can also be formulated and administered via gene guns or hydrogel catheters.
  • a combination of a recombinant adenovirus particle such as a recombinant adenovirus particle and an anti-SPO11 antibody
  • these may be used alone, but generally used together with a pharmaceutical substance.
  • the Such carriers already include the upper body, as well as water, saline, glucose, human albumin.
  • the number of administrations may be once a day.
  • the administration period may range from 1 day to several months or more, and the virus vector particles used in the present invention may be intermittently administered over a long period as 1 to several sets.
  • Kuta nucleic acid molecules can be used to diagnose specific cell and Z or tissue conditions. For example, it can be used for detecting and diagnosing tumor cells by incorporating a detectable marker gene into a viral molecule and combining it with the virus vector particle 1 antibody obtained by appropriate transfection. Alternatively, a detectable label on the anti-SP011 antibody can be used to detect and diagnose cells. 5. Cancer diagnostic agents and methods
  • the present invention also provides a diagnostic agent for cancer.
  • the diagnostic agent for cancer of the present invention comprises (a) an SP protein protein, or (b) a SP gene or a part of the base gene under hybridizing conditions. Contains a polynucleotide consisting of a hybrid sequence.
  • the antibody against SPO l 1 protein is SPO l 1
  • the binding of the SP 0 11 protein or the O 1 1 antibody is detected and / or quantified using the PO 11 antibody.
  • subject-derived biological sample includes subject-derived or body fluid (for example, blood (including whole blood, plasma, serum, etc.), saliva, sweat, semen, etc.).
  • a “subject” is a human subject who is, or is suspected to be, a human subject who is to receive, or wants to receive, an example of such a cancer is the large intestine. Cancer, stomach cancer, lung cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, cancer, uterine cancer (eg cervical cancer, uterine body cancer), testis knee Colorectal cancer including ovarian cancer, brain tumor, blood tumor, etc. is preferable.
  • the immunoassay for SP011 in biological samples derived from subjects as described above has the potential for cancer (eg, colon cancer).
  • One antibody is collected from a biological sample collected from a subject at risk for cancer. This includes measuring the amount of immunospecific binding between the anti-SP011 antibody and the contact antibody under conditions that cause binding. Body binding is used to detect the presence and expression of SP011 protein. In this case, the detection of increased SP 0 11 data is an indicator of the disease state.
  • biological test 1 The protein level should be the level of a healthy person who does not have cancer. taking measurement. Appropriate detection conditions for each measurement can be determined as appropriate by a conventional tester.
  • One method for detectably labeling anti-S PO 1 1 antibodies is to bind the body to an enzyme, eg, an enzyme such as the enzyme immunoassay (EIA) [Immunoadsorption assay with V o 1 1 er, A] ("T he Enzyme L inunosorbent A ssay) (ELISA), agnostic hormones, 2 l-7, Milogical A ssociates Quarte 1 ication, Walkersvi 1 le MD, A by JC lin Pathol, 3 1 5 0 7 9 7 8: Meth Enz 7 3 4 8 2 to 5 2 3, 1 9 8 1] by Butler, JE] Chemistry detected by fluorescence measurement by visible means by photometric measurement of binding to antibody React with appropriate and preferred substrates in such a way that the molecule is generated
  • Enzymes that can be labeled with a detectable label include, but are not limited to, peroxidase and alkaline.
  • This detection can also be achieved by colorimetric methods using chromogenic substrates.
  • Other methods that can be used in the present invention include racey (RIA), sandwich immunoassay, immunometry, fluorescence immunoassay (FIA), time-resolved fluorescence RFIA), enzyme immunoassay (EIA) ) Luminescence immunoassay Lines 35 to 20 (see page 47, line 47)).
  • various diseases related to the SP 0 1 1 tamper can be diagnosed by utilizing the in vivo quantification method of S protein using the antibody of the present invention. For example, if an increase in protein concentration is detected, for example, a diagnosis of a disease caused by overexpression of SP quality (eg, cancer (eg, large intestine • likely to be affected or in the future)
  • SP quality eg, cancer (eg, large intestine • likely to be affected or in the future
  • the anti-SP 0 11 antibody of the present invention can also be used in vivo, and is well known in the art for preparing antibody preparations that can be used here, for example, antibody-chelate uc 1 Med B iol
  • an antibody having a magnetic ion used in magnetic resonance imaging is described in, for example, Magnesonancein Medicine 1 9 9 1 2 3 4 2.
  • a probe or primer designed as a salt of the SPO 11 gene can be used.
  • the subject-derived SP011 gene or a fragment thereof can be used.
  • the base sequence used as a probe may be 12 bases or more, 15 bases or more, 18 bases or more, 21 bases or more, 27 bases or more, 30 bases or more, or a nucleotide fragment.
  • Hybridization Low, medium or high stringency conditions can be used.
  • SP ⁇ 1 1 A salt that can be highly pre-synthesized under the conditions of stripping to the base sequence of the gene or a fragment thereof.
  • this includes a Tsense polynucleotide complementary to the base sequence of the SPO11 gene or a fragment thereof.
  • the method of hybridization of phosphonic acid is known to those skilled in the art, and published in publication No. 8 90 6 6 98, EP-A 0 2 0 0 3 No. 2, 9 1 5, 0 8 2 EP—A 0 0 6 3 8 7 9, E 2 51, EP—A 0 1 2 8 0 1 8.
  • a known target sequence can be detected or quantified using a nucleotide probe or primer for the SP011 gene.
  • Southern hybridization Nose determination, RT-PCR method, PCR-SSCP method (Ge Vol. 5, pp. 874-8779 (1 989)) , ProceoftheNational Acad emy ofcesofthe United Statesolca, Vol. 8 6, 2 7 6 6-2 7 70 (1 9 8 9)
  • Method DNA chip or array C GH (C omp ara Cancer chip number abnormalities can be detected by simultaneously hydrating cancer-derived DNs labeled with different dyes using a chip on the genomic DNA fragments on the slide and detecting their binding status. This is a detection method with high resolution (P ineta 1 (1 9 9 8) N at G enet 2 0,
  • the mRNA of the SPO 11 cell housekeeping gene (eg, Shaper, JM ammary G land) B iole 3 (1 9 9 8) 3 1 5-3 2 4, Wu, YY and JL. Acta Derm Venereol 0) 2-3) mRNA levels, preferably RT-P it can.
  • the presence of a fragment or a fragment thereof in a test sample is detected using a mass spectrometer (MS). From the results of mass spectrometry, individual amino acids of proteins and peptides can be identified.
  • MS mass spectrometer
  • MAL D I matrix-assisted laser desonization
  • E I, C I electrospray ionization
  • FD field desorption
  • MAL DI matrix-assisted laser desonization
  • E I, C I electrospray ionization
  • FD field desorption
  • ion separation ion content compatible with ionization method
  • time-of-flight type ti me ght T0F
  • ESI time-of-flight type
  • ESI quadruple ion trap type
  • magnetic field Each type of mass spectrometer is used in tandem with a volume analyzer.
  • amino acid sequence determination by other amino acid sequence determination methods for example, one (eg, gas phase sequencer) may be used.
  • the present invention also provides a kit for Z or quantification using a subject SP011 protein or a fragment thereof containing an anti-SP011 antibody as a cancer marker.
  • the SP 0 1 gene or a fragment thereof in a body sample containing a base sequence that can be hybridized under stringent hypnotic conditions in the base sequence of SP or a part of SP is a cancer marker.
  • kits for quantification refers to a subject's body fluid (urine, lymph fluid, saliva, sweat, semen, etc.) or cells or normal tissue, or cancer cells or alternatively A molecule whose expression is enhanced, and that the presence of the molecule in the subject or tissue suggests the presence of cancer.
  • the kit of the first aspect described above contains a Z (quantitative component including SPO 11 protein and its partial peptide) in a body fluid sample from a subject.
  • a Z quantitative component including SPO 11 protein and its partial peptide
  • the antibody is labeled with radioactivity, fluorescence, colorimetry, or enzyme label.
  • the kit of the invention contains a labeled secondary antibody, and the kit according to the second aspect is based on a nucleotide sequence that can be hybridized under a stringent hybridization condition with the SP011 gene or sequence.
  • the kit of the invention containing the polynucleotide can comprise the above-mentioned poly immobilized on a DNA chip.
  • the kit of the present invention may be a container and a laser, in addition to the anti-SP01 antibody, SP011 residue, a base sequence that can be hybridized with a high-pridity hybridization. .
  • the label on or with the container contains a drug Example
  • Example 1 Colorectal cancer-specific amplification by Array C GH method
  • sample preparation and Array C verification were conducted for colon cancer specimens in 20 cases. .
  • Fig. 1 is a histogram showing the frequency of the SP 0 11 gene relative to the degree of amplification in colorectal cancer. Or the increase in the SP 0 11 gene in 200 colorectal cancer patients) and frequency. The average value indicates that the GZR value is 12 or more.
  • the SPOll gene was amplified in 650% of 20 0 patients, with an amplification degree of 7. The maximum value was 26, which was very frequent.
  • 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 with a high frequency in colon cancer patients.
  • C a co which is a cell line derived from colorectal cancer
  • Genomic DNA was extracted from the cultured cells according to the kit supplement using BLOOD & CELDNAKlt (QIAGEN).
  • Table 3 shows the R values of cell lines derived from colorectal cancer of the SP011 gene. As shown, it was found that amplification occurred in one gene located in the colorectal cancer-derived cell line AC C lone RP 1 1-6 7 1 P 16.
  • Table 4 shows cell lines derived from colon cancer of the SPO1 gene. Values are relative to control DNA (normal). It was also found that the SPO1 1 gene region exists in the colon cancer-derived cell line.
  • s 1 RN A synthesized specific s 1 RNA with specific 21m er in the gene (synthesized by Q I AGEN (Table 5)
  • L 1 pofec Invitrogen
  • N egatltro 1 sl RNA QI AGE N
  • the standard gene for calculating the relative ratio is G l y c e r a e — 3 — p h o s p h a t e d e h y d r o g e n a s H) C o n t r o l R e a g e n t s (A p p i e y s t e m s) is used to determine the expression level of GAPDH
  • the number of viable cells after introduction of s i RN A is measured by A 1 a m a r B l u e u r c e) according to the attached pro call and W a 1 2 0 M u l t l l a b e l z L um e n c e c e n c e r AR VO (P r k i n E l m e r)
  • SP 0 1 A 1 analysis was performed using RKO, a cell line derived from colorectal cancer.
  • Figure 2 shows that RKO cells have siRNA of SP011 gene.
  • Fig. 3 shows the results of measurement using the measurement reagent using the cells on the 4th day after s 1 RN fection of the S ⁇ 0 11 gene in RK 0 cells. The graph shows the relative amount to NC, and as a result of measuring the number of viable cells, the number of cells was significantly reduced compared to Ne Contro 1 (NC), as in the phenotype 3).
  • NC Ne Contro 1
  • Three s 1 RNAs of the SP 0 11 gene (a, b, c) were significant (P 0 0 1) in the t test.
  • RNA i effect significantly suppressed the growth of cancer cells in RKO as a result of the expression of the SPO11 gene.
  • Example 4 For RNA 1 analysis using a colon-derived normal cell line
  • the target gene inhibitory effect was cancer-specific verified by performing R using a cell line derived from a normal tissue of the large intestine.
  • RNA 1 derived from normal colon tissue of the SPO 11 gene are as follows.
  • FIG. 4 shows an observation image obtained on the 5th day after s i a n s f e c t i o n of S pO 11 gene in C CD 1 8 C O cells (lower ⁇ 2 0 0).
  • S P O l l c is a S P O l l gene species (c sequence of Example 3), and N C was almost the same as NC in the phenotypic observation as shown by a negative expression (FIG. 4).
  • FIG. 5 shows the results of measuring the measurement samples using the cells on the 5th day after the S p a l l s f e c t l l o n of the C p 18 C o cells.
  • the graph is relative to NC, and as with the phenotype, it was Negat 1 V e C o n t r o l (like, and no effect was observed (Fig. 5).
  • Figure 6 shows the results. As shown in the figure, a band around 2 Okb was found in the report of Shannon M et al (1999) FEBS Letters, 462, 329-334). This time, in addition to the 16 organs that were Shann, they were connected to the stomach (Stomach), the thyroid (Thy (Spinal Cord), the lymph node (Lymph Node), the trachea (Tra (Adrenal Gland)), and the bone marrow (Bone Marrow) However, for organs other than the testis, the expression level is low. The expression level is low in normal tissues, and it is testis-specific. result
  • Fig. 7 shows a part of the cancer cells observed in each specimen tissue (A ⁇ ; 0) (Analysis micrograph (fluorescence image). As shown, 3 or more cancer cancers were found. 10 specimens with (G / R) of 12 or more by the array CGH method were confirmed to have a SP range of 1. Also, it was confirmed that gene amplification occurred at the beginning of the disease stage. This indicates that the gene region can be applied not only as a molecular target for cancer therapeutics but also in cancer diagnosis Example 7 ⁇ SPO 1 1 protein 1 in blood by mass spectrometry 1) Pretreatment of blood samples
  • Peptide fragment 0 1 amount was separated by -Precolumn cartridge (C 5 DI, 300A, 300 zm idx 5 mm LC PACKINGS 163589) Nano column (C18 PepMap 3 u in, 100 A, 75 mid PACKINGS 160321) .
  • As the HPLC apparatus Ulti PACKINGS was used. The flow rate was 200nL / nnn, with a linear gradient of 0 57% / ⁇ with a gradient of 0 1% formic acid (Wak containing 2% acetonitrile (MERCK 1287229) and 0 1% formic acid tolyl).
  • the separated sample was introduced by ion trap mass spectrometry directly connected through PicoTip (New Object 10-D-20)
  • the mass spectrometer was ionized with HCT Plus (Bruker Dal tonics) material, with a capillary voltage of 1500 V, an end play of 500 V, and a dry gas.
  • the flow rate was 12 L / min, the dry gas temperature was 250 ° C.
  • the ion trap was set to perform MS / MS analysis with a Da capture around the target m / z.
  • RNAi analysis was performed using the above 3. s 1 RNA into cells was introduced into cells according to the attached protocol using nM siRNA (QI). 13% and 19% of siRNA c showed a growth inhibition effect (significant in t-test).
  • NC negative control siRNA (inhibition of growth rate and induction of cell death compared to Qiagen NC (partially confirmed.
  • a, b, and c are shown in Fig. 10a, si, respectively)
  • RNAb and si RN A c Corresponds to RNAb and si RN A c
  • sl RNA c showed fewer cell divisions and cell-like cells. The suppression was due to mitotic inhibition.
  • the present invention provides a therapeutic agent for cancer, a diagnostic agent, a diagnostic method, a kit used for the therapeutic method, and the like. Therefore, it is useful in the field of this invention 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 Ceased WO2007037538A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016192942A (ja) * 2015-04-01 2016-11-17 学校法人北里研究所 リン脂質ヒドロペルオキシド依存性細胞死の検出方法及びキット
CN109402169A (zh) * 2018-11-01 2019-03-01 湖南文理学院 一种spo11基因的敲除方法

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WO2002099046A2 (fr) * 2001-06-05 2002-12-12 Exelixis, Inc. Spo11 utilises comme modificateurs de la voie p53 et methodes d'utilisation associees
FR2833969A1 (fr) * 2001-12-20 2003-06-27 Ct Medico Chirurgical De Tronq Procede pour preparer des biopuces, de haute qualite controlee, les biopuces thematiques ou de criblage ainsi preparees, et leurs applications

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WO2002099046A2 (fr) * 2001-06-05 2002-12-12 Exelixis, Inc. Spo11 utilises comme modificateurs de la voie p53 et methodes d'utilisation associees
FR2833969A1 (fr) * 2001-12-20 2003-06-27 Ct Medico Chirurgical De Tronq Procede pour preparer des biopuces, de haute qualite controlee, les biopuces thematiques ou de criblage ainsi preparees, et leurs applications

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016192942A (ja) * 2015-04-01 2016-11-17 学校法人北里研究所 リン脂質ヒドロペルオキシド依存性細胞死の検出方法及びキット
CN109402169A (zh) * 2018-11-01 2019-03-01 湖南文理学院 一种spo11基因的敲除方法

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