WO2007037538A1

WO2007037538A1 - Therapeutic or diagnostic application of spo11 gene

Info

Publication number: WO2007037538A1
Application number: PCT/JP2006/320017
Authority: WO
Inventors: Shinichirou Niwa; Yasutaka Makino; Tomoki Ikuta; Kazuya Arai; Takayuki Shindou; Hiromichi Ogura
Original assignee: Link Genomics, Inc.
Priority date: 2005-09-30
Filing date: 2006-09-29
Publication date: 2007-04-05
Also published as: WO2007037538A9; JPWO2007037538A1

Abstract

It is intended to provide a therapeutic agent for cancer containing an expression inhibitor or activity inhibitor of SPO11 protein; a method for screening a compound which can be used as an active ingredient of such a therapeutic agent; an antibody against the SPO11 protein; a diagnostic agent for cancer/a diagnostic method for cancer using the antibody and the like.

Description

Specification

SP 0 1 1 Therapeutic or diagnostic use of gene

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 (cancers) 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. On the other hand, 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. By age group, 60s are in the order of 50s and 70s. 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. In addition, one (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

Under these circumstances, there is a need for drugs or methods that treat and / or diagnose cancer.

In particular, it is a therapeutic agent with high specificity for cancer.

In view of the situation as described above, 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.

(1) SPO 11 1 gene inhibitor (c) Deco for SPO11 gene or part thereof

(d) SP 0 1 1 gene variant that acts on the S P 0 1 1 gene or a part of it.

(e) a nucleic acid capable of specifically cleaving a transcript of the SPO11 gene,

and

(f) Compounds that inhibit transcription or inhibition of S P 0 1 1 gene (excluding the above nucleic acids)

(3) A substance that inhibits the activity of SP 0 11 protein, comprising a substance selected from the group consisting of:

(4) The S PO 1 1 protein activity inhibitor is

(a) an antibody against the SP0 1 1 protein,

(b) an SP011 protein variant having a dominant for the SP011 protein, and

(c) Compound that binds to the SPO1 protein (except for the above-mentioned variant)

Including a substance selected from the group consisting of

(5) The cancer therapeutic agent according to any one of (4), wherein the cancer is colorectal cancer or cervical cancer.

(6) Screening the S P〇 1 1 gene expression inhibitor,

(a) SP0 1 1 (a) The SPO 11 protein is contacted with the test compound

(b) a step of binding the SPO1 1 protein to the test compound; and

(c) A screening method comprising selecting a compound that binds to the SPO11 protein.

(8) The method according to (7), wherein the cancer is colorectal cancer or cervical cancer.

(9) An antibody against the SP0 1 1 protein.

(10) A cancer therapeutic agent comprising the antibody according to (9) above. (11) The above (10 therapeutic agent, further comprising a vector carrying a radioisotope, a therapeutic protein, a low molecular drug gene.

(1 2) The cancer treatment agent according to (1 1), wherein the cancer is colorectal cancer or cervical cancer.

(1 3) A cancer diagnostic agent comprising the antibody according to (9) above.

(14) 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.

(1 5) The cancer diagnostic agent according to (1 3) or the above, wherein the cancer is colon cancer.

(16) For cancer diagnosis containing the antibody according to (9) above

(1 7) SPO 1 1 gene or a part of its base sequence can be highly prehydidized under high hybridization conditions The method described in 9).

(2 1) SP 0 11 protein or quantified using a mass spectrometer, (2 2) SPO 1 1 using (2 9) anti-SPO 1 1 antibody described in (1 9) or (2 0) above Any one of the above (19) to (21) (2 3) (a) a step of contacting a biological sample derived from a subject with an antibody comprising SP011, and

(b) a step of detecting and Z or quantifying the antibody in the sample and the SP 0 11 tamper;

The diagnostic method according to (2 2) above, comprising:

(24) (a) A process comprising a polynucleotide consisting of a biological sample derived from a subject and a base sequence that is stringent to the base sequence of the SP011 residue fragment,

(b) detecting and detecting hybridization of the polynucleotide in the sample with SP or a fragment thereof,

The method according to (19) or (20) above, comprising:

(2 5) The method described in (19) to (24) above for use in diagnosis of cancer.

(2 6) 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

(3 2) 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. Brief Description of Drawings

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.

Figure 5 shows a cell line 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)) is involved in DNA double-strand formation and chromosome pairing during meiosis (Romanienko, PJ Otero, RD (1999) Genomics 61, 156-169, Shannon Occurs and recombination is a characteristic of sexual reproduction.

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). The lepto pairing DNA is aligned in the center and starts from the zygotene period, and the pachytene period is the period during which SC is formed. The SC disappears during this period, and the chromosome is decondensed during the deakinesis phase, when the chromosome is decondensed and the transcription is active.

S C has a protein structure called Central element (CE) and its side element (LE) in the center. Scp3 of LE (Dobson, MJ, et al (1994) J Cell Sci 107, known. 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

Genes involved in homologous recombination during meiosis are considered to be living (Walker, MY & Hawley, Chromosoma 109, 3-9). Discovered by research in yeast, Mouse (Keeney, S, et al (1999) Genomics Romanienko, PJ & Camer lni-Otero, RD (1999) 156-169, Shannon, M, et al (1999) FEBS Lett 4 Metzler-Guilleman, C & de Massy, B (2000) Chr 133-138), human (Romanienko, PJ & Camerin (1999) Genomics 61, 156-169, Shannon, M, et al Lett 462, 329-334) This is an example of the fact that homologous recombination during meiosis is in a common system between species.

SPO l 1 is homologous to the subunit of Top (T0P6A), which 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 & Erenpreisa, J (2005) Cancer Cell Internatio 11) Traditionally used as a serum antigen for colorectal cancer (2002) Cancer Res 62, 6750-6755)

SP011 directly acts on DSBs during meiosis (Neale, (2005) Nature 436, 1053-1057). In recent years DSBs formation has been considered an important step in cells. Cells Abnormal stimuli become stress during DNA replication, and in some cases, DSBs are generated. They are subject to DNA damage check repair and apoptosis growth inhibition. However, if CheckBoin 機能 does not work, such as when In addition, the formation of DSBs causes chromosomal instability and contributes to the progression (Gorgoulis, V G, et al

434, 907-913) The expression of SP011 in ₀ cancer may have a role in cancer.

Also known as a typical anti-cancer agent,

(Topoisomerase I inhibitor) and Doxorubicin (, Τορο lsomeras increase cancer cells by inhibiting 卜 poisomerase activity. S Ρ〇 1 1 is classified as the same DNA Top as Topoisomerase I and Π. 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. In addition, SP 0 1 1 genetics And (2) SP 0 1 1 TAN A cancer therapeutic agent containing a harmful substance as an active ingredient.

In the present specification, the term “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. From the polynucleotide consisting of a base sequence that can be hybridized and re-synthesized to the base-complementary sequence of the relevant gene, such as a mutant that is used in the specification "SP 0 1 gene" The hyperprecipitation included in the method is a known method or a method described in, for example, Molecular Cloning (Moleculalng Third Edition, JS ambra 1, Old Spring Harbor Labs 2 0 0 1). Commercially available library When using, it is possible to perform thus in the attached instructions. Here, “stringent stringent conditions, medium stringent conditions, or stringent conditions can be used.“ Low stringent conditions include 5 XSSC, 5 X Denhardt's solution, 0 5% SDS, 32 ° C. Also, “Medium stringer” There are several factors that affect the temperature, probe concentration, long probe time, salt concentration, etc., and those skilled in the art can achieve the same stringency by selecting them as appropriate.

As a hybridizable polynucleotide, 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.

In the present specification, “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.

In this specification, “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. In general, the higher the homology value is, the 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. For example, 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. Preferably, the amino acid corresponding to the part involved in the activity of these polypeptides. Further, 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). The base sequence is resolved using BLAS TN. For example, score = 100 and wordl 12 are used for lame. The amino acid sequence using BLASTX is no. For example, score = 50 and wo rd = 3 for lame. When using B LA ST and G apped BLAST, use the default parameters of each program. In this specification, the term “cancer therapeutic agent” refers to an anti-cancer agent, In one embodiment, the present invention provides a cancer therapeutic agent containing SPO 11 gene as an active ingredient.

In the present specification, there is no limitation as long as it inhibits gene expression in the “SP〇1 1 gene expression inhibitor”, but examples include inhibiting transcription from the P0 1 gene to SP 0 1 1 mRNA. And (ii) a substance that inhibits SPOll from SP0ll mRNA.

As an example of transcription from S P 0 1 1 gene to S P 0 1 1 mRNA,

(a) Anti-S P 0 1 1 gene or part thereof

(b) Decoy for S P O 1 1 gene or part thereof

(c) SPO1 1 gene variant acting on the S P O 1 1 gene or a part thereof, or

(d) Other transcription inhibitor compounds

Etc. are included.

In addition, examples of substances that harm SP0 1 1 protein from S PO l l mRNA include:

(e) a polynucleotide that is R to S P O l l mRNA or a portion thereof (eg, s 1 R N A),

(f) an nucleotide for S P O l l mRNA or a part thereof,

(g) 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.

In the cancer therapeutic agent of the present invention, a nucleic acid having an action of inhibiting by the effect of the SP 0 11 gene can be used as an active ingredient. 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. Such 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 Such 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

A c i d s R e s, May 15, etc.).

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. Specifically, the following s 1 RNA (used in Example 3) was used (Table 1).

In the present specification, the term “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.

For example, if 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.

Effective expression of target gene using antisense nucleic acid 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. As used herein, “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). For hammerhead ribozymes, 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, ρ 2 1 9 1; Nuc 1 A cids R 1 7, p 7 0 5 9 etc. can be referred. For bosaim, for example, 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.

Furthermore, in the present invention, a compound that inhibits the transcription activity of the SP 0 11 gene can be used as an active ingredient. For example, 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,

(b) a S P 0 1 1 protein variant having a dominant negative relative to the S P 0 1 1 protein, or

(c) Compounds that bind to SPO1 1 protein (excluding the above)

Etc. are included.

As used herein, “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 (anti-SP011 antibody) can be prepared publicly by those skilled in the art. The anti-SP011 antibody will be described later. -

In the present specification, “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

(See Kunihiro Tsuchida, Gene Activity Inhibition Experiment, edited by Kazumasa Tahira, 0 1) 2 6-3 2).

Furthermore, in the present invention, as a substance having SP011 protein, a compound other than the above-mentioned compound that binds to SP011 protein can be used as an active ingredient. 2. SP〇 1 1 Inhibits protein activity or expression Leaning method

The present invention also provides a screen for a candidate compound having a cancer suppressing action.

One preferred embodiment is a method using S P O 1 1 protein and a test as indicators. In general, SPO11 protein has an effect of inhibiting the activity of SPO1 protein. Here, it is preferable that the compound is an SPO11 protein. In this method, first, 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.

Next, in this method, binding to the SP011 protein is detected.

There is no restriction | limiting in particular as a test compound used for this method. Compounds, organic compounds, inorganic compounds, proteins, peptide compounds, and compound libraries, gene libraries, cell extracts, cell culture supernatants, fermented microorganism products, marine extracts, etc. . II Measurement of topoisomerase activity (Bergerat, A et Biol Chein 269, 27663-27669)).

Next, in this method, a test compound that inhibits sex is selected.

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.

In this method, first, a compound that expresses the SP011 gene is contacted. Examples of the origin of “cells” used include, but are not limited to, pet, and house cells such as Su, Cat, Inu, Hushi, Hedge, Tri. As the “cell expressing SP”, 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. Here, in the “gene expression”, the expression levels of transcriptional and translational genes can be measured by methods known to those skilled in the art. For example, 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. Alternatively, 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. It is also possible to measure the translation level by collecting protein fractions from SP011 cells and detecting the SP011 data by electrophoresis such as SDS-PAGE. Furthermore, it is possible to determine the gene by detecting the expression of the protein by Western blotting using an antibody against SP011. There are no particular limitations on SP 0 1 1 protein detection as long as it is a detectable antibody, but both oral and polyclonal antibodies are used.

In this method, 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.

Used for cancer targeted therapy or targeted drug delivery

3, 1 Anti-SP0 1 1 antibody

In the present specification, “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. In addition, “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). )

(1) Preparation of antigen

In the present invention, it 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. Examples of 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.

(2) Monoclonal anti-SPO1 protein

(1) Collection of antibody-producing cells

SP0 1 1 protein as above, its partial peptid

(In the present specification, in the description of the antibody, these are collectively referred to as 1 protein.) As an antigen and administered to a mammal such as a mouse or a rabbit. When the antigen per animal is not used, 0 1 to: L 0,0 mg, and when used, 1 to 100 g. Examples of the adjuvant include complete adjuvant (FCA), incomplete Freund A), and aluminum hydroxide adjuvant. 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. Examples of antibody-producing cells 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. Examples of 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. In addition, antibody-producing cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electon).

(ill) Selection and cloning of hybridomas As a method of target hybridomas from cells after cell fusion treatment, the cell suspension is appropriately diluted with, for example, RP 0 medium containing urchin fetal serum, and then microtiter is used. Plate about Zwe 1 1 and add selective medium to each well, and replace the ground. As a result, obtain cells that grow after the culture is started in the selective medium as a hybridoma. Next, determine whether antibodies that react with S protein are present in the culture supernatant of the hybridoma that has proliferated. Screeninin Stand up.

(i v) Monoclonal antibody collection

As a method for monochromating from the hybridoma obtained as described above, a normal cell culture method or ascites formation method can be used. In the cell culture method, 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.

(3) Polyclonal anti-antibody against SPO 11 protein First, 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. Examples of 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.

Next, for example, 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.

(4) Antibody fragments

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 〇 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. Among the most commonly used fluorescent labeling compounds, 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.

In addition, 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. When used in combination with a drug, 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.

In the present invention, 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. For example, the ^{1 25} I or ¹ de reduction by known methods of chloramine-T method, can be antibodies or binding. In addition, tech m for diagnosis, 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. As 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.

In the present invention, as an example of the “therapeutic protein”, site force-in that activates is suitable. For example, human 2, human granulocyte-macrophage colony-stimulating factor, and colony-stimulating factor Human interleukin 1 2 etc. In addition, ricin and diph toxins can be used to directly kill colon cancer cells. For example, therapeutic protein It may mean a diagnostic or therapeutic compound other than “protein”. Examples of “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.) For example, 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 daunomycin and the carboxyl group of the antibody using water-soluble carbohydrate, and the like.

As an example of “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〇1 1 antibody Injected to patients who need gene therapy together with SP〇1 1 antibody Antigen recognized by anti-SP〇1 1 antibody (ie, can be targeted to SP011 site) .

The anti-SP011 antibody and the other drug can be chemically or conjugated. Here, “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.

4. Formulation and administration method

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.

In formulating the therapeutic agent of the present invention, according to a conventional method, 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. For example, 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. In the case of oral administration, about 0 l mg to l per day for general (as 60 kg) Preferably about 10 to: L 0 mg, more preferably about 1. When administered parenterally, 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) However, it is preferable to administer about 0 1 to 20 mg, preferably about 0 1 to 1 O mg by intravenous injection. However, ultimately, the type of dosage form, administration method, disease 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.) Preferably, 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. In addition, the agent of the present invention may contain both the SP 0 1 protein activity inhibitor PO 1 1 gene expression inhibitor. In the therapeutic agent of the present invention, 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.

In the present invention, when a combination of a recombinant adenovirus particle, such as a recombinant adenovirus particle and an anti-SPO11 antibody, is used for cancer treatment, 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. In general, it is preferable to administer one virus particle at a time per adult, but this may vary depending on the nature of the disease state. 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. Preferably, 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.

5.1 Diagnostic agents and diagnostic methods using anti-SP0 1 1 antibodies

The antibody against SPO l 1 protein is SPO l 1 Alternatively, in the above-described detection and Z or quantification step, the binding of the SP 0 11 protein or the O 1 1 antibody is detected and / or quantified using the PO 11 antibody.

In the present specification, “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. If necessary, 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)).

As described above, 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) It should be noted that 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 In addition, 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.

5.2 Polynucleotide (eg, DNA) pro-blocking and diagnostic methods

In the diagnostic method of the present invention, a probe or primer designed as a salt of the SPO 11 gene can be used. For example, (a) the subject-derived SP011 gene or a fragment thereof can be used. Bases that can be hyper-predated under string pre-conditioning conditions And determine or quantify. 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〇 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.

It is also assumed that 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.

In the diagnostic method of the present invention, a known target sequence can be detected or quantified using a nucleotide probe or primer for the SP011 gene. Thus, for example, 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,

1) o

In the present invention, in order to detect whether or not the SP 0 11 gene is expressed, 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.

In the target sequence (disease caused by overexpression of SP〇1 1 (DNA, mRN output and quantification and overexpression of SP〇1 1 gene was confirmed by the above method) (for example, cancer) It is likely that there is a possibility, or it can be affected in the future.

5.3 Diagnosis method using mass spectrometer

In another embodiment of the diagnostic method of the present invention, 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.

There are various types of ionization, such as matrix-assisted laser desonization (MAL D I), electrospray ionization, phase (E I, C I), and field desorption (FD). For ion separation, ion content compatible with ionization method For example, in case of MAL DI, time-of-flight type (ti me ght T0F) mass spectrometer, in case of ESI, quadruple ion trap type, magnetic field Each type of mass spectrometer is used in tandem with a volume analyzer. For example, I MSZMS, Q—TºF MS, MALD I—TºF. In addition, amino acid sequence determination by other amino acid sequence determination methods, for example, one (eg, gas phase sequencer) may be used.

5.4 Diagnostic kit

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. In addition, 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. Also provide kits for quantification. this In the present specification, the term “cancer marker” 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. For example, detection and / or quantification by SP01 ELISA This can be used to detect and Z or quantify levels in, for example, tissue sections, or body fluid samples such as blood or urine.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 In this example, sample preparation and Array C verification were conducted for colon cancer specimens in 20 cases. .

As a result, amplification occurred frequently in colorectal cancer specimens, and gene information in the public DB (NC BI: http // wn 1 mnihgov /) was used, and as a result of careful examination, C lone RP 1 1— 6 7 1 P SPO 1 1 meiotic proteincovaboundto DSB — 1 ike (S cere)) (NCBIA ccession No NM 4) gene located at 1 6 is frequently and highly elevated in colorectal cancer patients (Figure 1, Table 2) . 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.

As shown in Table 2, 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 In this example, the amplification in a colon cancer-derived cultured cell line was verified with a high frequency in colon cancer patients.

Sample preparation>

As a cultured cell line, C a co, which is a cell line derived from colorectal cancer, was used. Genomic DNA was extracted from the cultured cells according to the kit supplement using BLOOD & CELDNAKlt (QIAGEN).

In order to confirm the amplification of the gene region, the details of the array GH were carried out in the same manner as in Example 1. Result

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 3)

<Quantitative PC R> It was. Primers are synthesized with the following sequences (○ PE RON use / this.

Primer sequence

5 '— T TT G CAG C TGAT G G C TTGT G — 3 3)

5 '-AAC GGTTGAC TTGGCAGC TATT Column number 4) Result

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.

(Table 4)

Based on these results, cell lines derived from colorectal cancer (SP 0 11 gene was confirmed to be amplified even in Caco, and could be used for functional analysis of S PO 11 gene in cancer. . RNA i analysis

Cell lines were purchased from ATCC and attached to the protocol. s 1 RN A synthesized specific s 1 RNA with specific 21m er in the gene (synthesized by Q I AGEN (Table 5)

For introduction of sl RNA into RK0 cells, L 1 pofec (Invitrogen) was used, and it was introduced into the cells according to 50 nM si RN port. N egatltro 1 sl RNA (QI AGE N) was used as a control. Observation was performed under an inverted microscope.

Use a quantitative RT_PCR method to verify the effect of siRNA with. s 1 From the cells 24 hours after RNA transfer, use M i -M idi Total RNA Purification (Invitrogen) Use (A pplled B iosyste rn s) and follow the call to 7 5 0 0 Real-Time PCR

Using (A p p l l e d B i o s y s t e rn s), the realmer synthesized the following sequence (commissioned to 〇 P E RON) Primer sequence ·

5,-G G C C T C C AGT T C T GAG GT T C T No. 1 1)

5 '-T T C C C A G C T T GA T C T G T T G T C T (SEQ ID NO: 1 2)

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 Ρ 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). Three s 1 RNAs of the SP 0 11 gene (a, b, c) were significant (P 0 0 1) in the t test.

Therefore, the 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 In this example, 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.

For the cell line, use CD 18 CO purchased from AT CC, and follow the attached protocol. The sl RNA c sequence used was used. The siRNA was introduced into the cells using Liamine 200 (Invitrogen) and siRNA was introduced into the cells according to the attached protocol. gatlve ControlsiRNA (QI AG E) The cells were observed under an inverted microscope for 5 days after introduction. The effects of the cell line CC 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).

As a result, the RKO effect, which is a colorectal cancer-derived cell line, suppressed the expression of the SP 0 11 gene, and as a result, the growth of was suppressed, but the normal colorectal tissue-derived cell line CCD had no effect. It was found. Therefore, we found a cancer-specific target genetic for drug discovery because it has little effect on S PO l l inheritance. Example 5 Evaluation of organ specificity

What is the organ-specific nature of SP 0 1 1 gene in normal tissues? Northern hybrids known in the art (Lung), Liver, Skeletal Mus (Kidney), Knee (Pancreas), Spleen (Spleen), Thymus (Prostate), Testis, Ovary (Intestine), Colon (Colon), Per Leukocyte, Stomach, Thyroid, Cord, Lymph Node, Trachea, Gland, Bone Marrow) The Yon method was used for analysis. As mentioned above, SP 0 1 1 is the result of NCBI (ht tp // www ncbi nlm nih gov; registering 1, 826 bp of A NM_012444 as mRNA.

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

Serum specimen of colorectal cancer patient 10 L and healthy subject serum specimen 10 ^ L F solution UOmM Tris HC1 ph 74 4 + 150 mM NaCl) Albumin using 500 ProteomeLab IgY-12 SC proteome partition (BECKMAN COULTER A24618) A large amount of protein was removed from the globules. A final concentration of 10 mM dithiothreitol (Wako 049-08972) was added to the obtained fraction, and a reduction reaction was performed. After the reaction was completed, a final concentration of 10 mM was added (SIGMA 144-48-9), and the alkylation reaction was carried out at room temperature. After completion of the reaction, add 4 times the amount of cold acetate (08681, -20 ° C) and leave it at 80 ° C for 1 hour. 2) Analysis using Nano-HPLC direct mass spectrometer

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% / ιηιη 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.

3) Data analysis

All analysis data obtained from the mass spectrometer was output via Data Analysis (Bruker Daltonics). Only the analysis data of all demolecules that were output were extracted. In the extracted data, we examined the MS / MS data and analyzed the presence or absence of an ion peak with a mass that fragments the target molecule. As shown in Fig. 8 and Fig. 9, the ion peak corresponding to the flag of the target molecule was found in the serum from colon cancer patients. That is, as shown in FIG. 9B, at least a partial sequence called C-terminal PAE is determined, which is amino at positions 66 to 72 in the SP011 mino acid sequence (SEQ ID NO: 2). Therefore, as shown in FIG. 9A, the amino acid sequence 66-position peptide fragment (SEQ ID NO: 13) was identified. The ion peak corresponding to the sequence was not analyzed using normal serum (see Figure 9C). Therefore, it was suggested that this target molecule (Sun 1 protein) increased in the blood abundance specifically for cancer. Example 8. RNAi effect in cervical cancer-derived strain HeLa cell line In colon cancer cell line, SPO 11 gene knockdown suppression effect was observed, but in cervical cancer cell line Evaluation was made by the RNAi analysis method described above.

Using the HeLa cell line of cervical cancer-derived cells, 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).

Furthermore, following the time series, differential interference images were taken under a microscope and observed in detail. After transducing siRNA (a, b, c) into HeLa cells, differential interference images in the same field were observed 1, 2, 3, and 4 days later. 1 Shown in 1. NC is negative control siRNA (inhibition of growth rate and induction of cell death compared to Qiagen NC (partially confirmed. In the figure, a, b, and c are shown in Fig. 10a, si, respectively) Corresponds to RNAb and si RN A c As shown in Fig. 1, induction of cell death in b where growth inhibition was observed In addition, sl RNA c showed fewer cell divisions and cell-like cells. The suppression was due to mitotic inhibition.

This result suggests that inhibition of S P 0 1 1 gene function suggests that the large intestine has an antitumor effect even in cervical cancer Industrial applicability

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.

Claims

The scope of the claims

1 S P O l l gene expression inhibitory substance as an active ingredient.

2 The expression inhibitor of the SP0 1 1 gene is

(a) a nucleic acid having the expression of the SPO1 1 gene by the RNA ι effect,

(b) a nucleic acid in the transcription product or a part thereof of the SPOll gene,

and

(c) a nucleic acid capable of specifically cleaving a transcript of the SPO11 gene,

The therapeutic agent for 3 S P O 1 1 protein activity inhibitor according to claim 1, comprising a substance selected from the group consisting of:

4 The activity inhibitor of the SP0 1 1 protein is

An antibody against the protein,

The cancer therapeutic agent of Claim 3 containing this.

5 The cancer therapeutic agent according to any one of claims 1 to 3, wherein the cancer is colorectal cancer or cervical cancer.

6 S P〇 1 1

(a) SP0 1 1 (a) Contact SPO 1 1 protein with test compound

(b) a step of binding the SPO1 1 protein to the test compound; and

8. The method according to claim 1, wherein the cancer is colorectal cancer or cervical cancer.

9 S P〇 1 1 Antibody against protein.

10. A cancer therapeutic agent comprising the antibody according to claim 9.

11. The therapeutic agent according to claim 10, further comprising a vector carrying a radioisotope, a therapeutic protein, a low molecular drug, and a gene.

1 2 The cancer therapeutic agent according to claim 11, wherein the cancer is colorectal cancer or cervical cancer.

1 3 A cancer diagnostic agent comprising the antibody according to claim 9.

1 4 SP 0 11 A diagnostic agent for cancer that can be highly pre-polymerized under high pre-hybridization conditions in one gene or a part of its base sequence.

1 5 The diagnostic agent according to claim 1, wherein the cancer is colon cancer.

1 6 Cancer diagnosis kit containing the antibody according to claim 9.

1 7 SP ○ 1 Can be hyper-predated to a single gene or a part of its base sequence under high pre-hybridization conditions The method described.

21. The method according to claim 19 or 20, wherein the SP 0 1 1 protein is quantified using a mass spectrometer.

2 2 Anti-SP 0 1 1 antibody is used, S P 0 1 1 protein Z or quantified, The method according to any one of claims 19 to 21

2 3 (a) a step of contacting a biological sample derived from a subject with an SP1 1 1 antibody, and

(b) a step of detecting and / or quantifying the antibody in the sample and the SP 0 1 1 tamper;

The method according to claim 22, comprising:

24 (a) A polynucleotide process comprising a biological sample derived from a subject and a base sequence that can be hybridized in a stringent manner to the base sequence of the SP 0 1 1 genetic fragment, and

(b) detecting and detecting the hybridization between the polynucleotide in the sample and SP or a fragment thereof;

The method according to claim 19 or 20, comprising:

25. A method according to claims 19 to 24 for use in diagnosis of cancer.

2 6 The cancer according to claim 25, wherein the cancer is colorectal cancer. 2 7 Polynucleotide having the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10