WO2008072676A1

WO2008072676A1 - Novel disease marker and diagnosis using the same

Info

Publication number: WO2008072676A1
Application number: PCT/JP2007/073974
Authority: WO
Inventors: Teruto Hashiguchi; Shoji Natsugoe; Ikuro Maruyama; Kenji Tanaka; Lyang-Ja Lee
Original assignee: Kagoshima University; Protosera Inc.
Priority date: 2006-12-12
Filing date: 2007-12-12
Publication date: 2008-06-19
Also published as: JPWO2008072676A1

Abstract

Disclosed is a test method for diagnosing pancreatic cancer, breast cancer, uterine corpus cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroid, endometriosis or Crow-Fukase syndrome in a subject, which is characterized by determining the quantity of a peptide comprising the amino acid sequence depicted in SEQ ID NO:1 (preferably by mass spectrometry or by using an antibody recognizing specifically the peptide) in a biological sample (preferably a body fluid, more preferably a serum, a plasma or the like) collected from the subject.

Description

Specification

Novel disease markers and diagnosis using them

Technical field

[0001] The present invention relates to various cancers including presumptive cancer and non-cancerous diseases such as uterine fibroids and endometriosis, as well as novel diagnostic markers for the early stage of Crow's Fukase syndrome, and the diseases using the same. It relates to diagnostic methods.

Background art

[0002] When thrombin acts on fibrinogen, it first hydrolyzes between the 16th Arg and 17th Gly from the N terminus of the A α chain to cut out fibrinopeptide A. Next, fibrinopeptide B is cut out between the 14th Arg and 15th Gly from the Ν end of the B β chain. The function of these excised fibrinopeptides is not well understood, but their presence in the blood is evidence that hypercoagulation is occurring in the blood vessels. Therefore, measurement of blood fibrinopeptide is used as an indicator of blood coagulation in blood vessels. The RIA method is used for clinical fibrinopeptide measurements.

[0003] Recently, mass spectrometry was used to examine the pattern of proteolysis products in the sera of several cancer patients, and fibrinopeptide A (hereinafter referred to as FPA) or fragments thereof were found in prostate cancer, bladder It has been reported that it varies with cancer and breast cancer (Non-patent Document 1). In particular, a peptide consisting of 15 amino acid residues lacking the N-terminal Ala residue of fibrinopeptide A is described as significantly lower in the serum of prostate and bladder cancer patients, but unchanged in breast cancer patients. ing. However, there has been a report on the relationship between FPA or its fragments and presumptive cancer V /!

[0004] Crow'Fukase syndrome (hereinafter also referred to as CFS) is known as an intractable disease with a poor prognosis. The inventors previously identified a variety of relatively low molecular weight peptides that appear in the blood as the disease progresses in a series of processes from CFS diagnosis to death outcome. I have applied. These peptides are not limited to CFS, but are derived from a wide variety of underlying diseases, and as a result of the degradation of proteins present in the living body, functional and organic disorders have developed (the present inventors It appears in common with the pathological condition is called “biodegradation syndrome” The peptides that vary specifically in the early stages of CFS have not yet been clarified.

Non-Patent Document 1: Villanueva et al., "The Journal of Clinical Investigation J, Vol. 116 (No. 1), p. 27 1-" 284 (2006)

Disclosure of the invention

Problems to be solved by the invention

[0005] An object of the present invention is to provide a novel diagnostic marker for cancer and early CFS, and an effective diagnostic method for the disease using the same.

Means for solving the problem

[0006] As a result of mass spectrometry analysis of sera collected from various cancer patients and CFS patients who should achieve the above-mentioned objectives, the present inventors have found that in presumptive cancer patients, compared with normal subjects, A peptide having an increasing molecular weight of about 1466 was found. We also found a peptide with a molecular weight of about 1466, which is markedly elevated in patients with CFS in the definite phase. Analysis of the amino acid sequence revealed that these peptides were identical peptides consisting of 15 amino acid residues lacking the N-terminal Ala residue of FPA. Surprisingly, this peptide was also found to be significantly elevated in cancers other than prostate cancer and bladder cancer that have been reported so far. In any case, breast cancer! /, Which showed a marked increase compared to prostate cancer, in any case, the peptide was identified as a diagnostic marker for these diseases because it was markedly elevated in presumptive cancer and early CFS. Thus, the present invention has been completed.

That is, the present invention provides:

[1] Victory cancer, breast cancer, endometrial cancer, ovarian cancer, child in the subject characterized by measuring the amount of peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in a biological sample collected from the subject A test method for the diagnosis of a disease selected from the group consisting of cervical cancer, uterine fibroids, endometriosis and Crow-Fukase syndrome;

[2] The method according to [1] above, wherein the biological sample is a body fluid;

[3] Body fluid is blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, The method according to [2] above, which is selected from the group consisting of aqueous humor, vitreous humor and lymph; [4] The method according to [1] to [3] above, comprising subjecting a biological sample to mass spectrometry. The method described in any one of the above;

[5] The method according to any one of [1] to [3] above, wherein an antibody that specifically recognizes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is used;

[6] The above-mentioned [1] to [5], wherein a biological sample is collected from a patient in time series, and the time course of the amount of peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in the sample is examined. ]! /, The method as described in somewhere;

[7] Evaluation of therapeutic effects in patients with diseases selected from the group consisting of presumptive cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, uterine fibroids, endometriosis, and Claude Fukase syndrome The method is characterized in that a biological sample is collected from the patient before and after treatment, and the change in the amount of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sample is examined. Method;

Etc.

The invention's effect

[0008] According to the present invention, various cancers including presumptive cancer, non-cancerous diseases such as uterine fibroids and endometriosis, and Crow's Fukase syndrome can be quickly and accurately determined. Early detection and early treatment are possible.

Brief Description of Drawings

FIG. 1 is a graph showing the distribution of the peptide peak intensity at the clinical stage of CFS patients.

FIG. 2 is a graph showing the distribution of the peak intensity of the peptide of the present invention in various cancer patients. Each point in the figure represents the relative peak intensity of the peptide of the present invention contained in the subject when the serum of one patient was measured four times. The horizontal bar indicates the average value of the relative peak intensities of the peptide of the present invention in each disease. The numbers in the figure show the T-test results (P value) of the relative peak intensity of the peptide of the present invention between vaginal cancer, other cancers, and healthy subjects.

FIG. 3 is a graph showing the distribution of the peak intensity of the peptide of the present invention at the clinical stage of a presumptive cancer patient. Each point in the figure represents the relative peak intensity of the peptide of the present invention contained in the subject when the serum of one patient was measured four times. The horizontal bar shows the peptide of the present invention The average value of relative peak intensity is shown.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The present invention provides a novel and useful diagnostic marker peptide (hereinafter sometimes referred to as "the peptide of the present invention") of presumptive cancer or CFS. The peptide of the present invention consists of the amino acid sequence shown in SEQ ID NO: 1. The peptide corresponds to a fragment from the second Asp residue to the 16th Arg residue from the N-terminus of the mature fibrinogen A α chain.

It is well known that fibrinopeptide A (FPA) is produced by cleaving between the 16th Arg residue and the 17th Gly residue from the N-terminus of fibrinogen A α chain by thrombin. In addition, a peptide having the same amino acid sequence as that of the peptide of the present invention lacking the N-terminal Ala residue of FPA has been reported (Non-Patent Document 1 above), and the serum level of the peptide has been reported for prostate cancer and It is described that there is a significant decrease in the serum of bladder cancer patients, and that there is no significant difference in healthy breast cancer patients. The present inventors have found for the first time that this peptide is markedly elevated in patients with presumptive cancer. Furthermore, it was unexpectedly found that the peptide was significantly increased! / In patients with CFS who had an intractable disease with a poor prognosis! /.

Further, the peptide of the present invention can be used for prostate cancer patients, breast cancer patients, ovarian cancer patients, endometrial cancer patients, cervical cancer patients, endometriosis patients, and cervical myomas in addition to patients with presumptive cancer and CFS. It is also significantly increased in patients. Therefore, the peptide of the present invention can also be used as a diagnostic marker for cancerous diseases and non-cancerous diseases including these cancers. In any case, the elevation of the peptide in presumptive cancer and early CFS is striking and is a very useful diagnostic marker for these two diseases.

[0011] As described above, the peptide of the present invention is a fragment (degradation product) of the polypeptide consisting of the 2nd to 16th amino acids from the N-terminus of the mature fibrinogen A α chain. Therefore, polymorphisms or alleles containing one or more amino acid substitutions, deletions, insertions or additions, or combinations thereof within the partial amino acid sequence of fibrinogen A α to be tested. It is obvious to those skilled in the art that when having a mutation, the amino acid sequence of the peptide to be detected should be interpreted as “the amino acid sequence having the polymorphism or allelic variation in the amino acid sequence shown in SEQ ID NO: 1.” I will.

[0012] The present invention also provides spleen cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine muscle. By measuring the amount of the peptide of the present invention in a biological sample derived from a patient suspected of having a tumor, endometriosis or CFS, the patient has premature cancer, breast cancer, endometrial cancer, ovarian cancer Provides a diagnostic method for the diagnosis of cervical cancer, prostate cancer, uterine fibroids, endometriosis or CFS. Here, “test for diagnosis” means measurement of the amount of the peptide and, if necessary, comparison with the measured value in a control sample. `` Patient cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis or patients suspected of having CFS '' are subjectively suspected by the patient. Although it may be based on some objective basis, it is preferable that the results of a conventionally known clinical examination and / or examination show that there is a reasonable possibility of such diseases. It is a patient who is judged by a doctor to be, or a human with an equivalent medical condition.

In the treatment of cancer and CFS, early detection and early treatment are the major principles. However, the peptide of the present invention has a definite CFS (an abnormal sensation or edema of the lower limbs, skin abnormality, organ enlargement, blood monoclonal protein). It is very significant in that it is a specific marker peptide at the time when endocrine abnormalities appear simultaneously or individually but the cause cannot be specified. The viscera is also called a “silent organ”, and early detection of visceral cancer is difficult compared to cancers of other organs, so by adopting periodic diagnosis using the peptide of the present invention as an index, Early detection of presumptive cancer · It may contribute to early treatment.

[0013] The patient-derived biological sample to be the test sample is not particularly limited! /, But it is preferable that the patient is less invasive, for example, blood, plasma, serum, saliva, urine, tears Examples include those that can be easily collected from living organisms, and those that can be collected relatively easily such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, and vitreous humor.

When using serum or plasma, it can be prepared by collecting blood from a patient according to a conventional method and separating the liquid component. The peptide of the present invention as a detection target can be separated and removed in advance using a spin column or the like, if necessary, such as a high molecular weight protein fraction.

[0014] The detection of the peptide of the present invention in a biological sample can be performed, for example, by measuring various molecular weights of the biological sample, for example, gel electrophoresis or various separation and purification methods (eg, ion exchange chromatography, hydrophobicity). Chromatography, affinity chromatography, reverse phase chromatography Ionization methods (eg, electron impact ionization method, field desorption method, secondary ionization method, fast atom collision method, matrix-assisted laser desorption ionization (MALDI) method, electrospray ionization method, etc.), mass spectrometry (E.g. double convergence mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, etc.) Forces that can be performed by detecting matching bands or spots, or peaks, but are not limited to these. Since the peptide of the present invention has a known amino acid sequence, a method of preparing an antibody that recognizes the amino acid sequence and detecting the peptide by Western blotting or various immunoassays can be used more preferably. Furthermore, the hybrid detection method described above is also effective.

[0015] The peptides of the present invention have a molecular weight of about 1465.5 to about 1465.9. Here, "Molecular weight approx.

`` '' Takes into account the range of error of each measurement method.For example, in the case of a method using a mass spectrometer, ± 0.5% (preferably ± 0.3%, more preferably It is preferable to measure the peak intensity that appears at the position of ± 0.1%! /.

[0016] One of the particularly preferable measurement methods in the inspection method of the present invention is that a test sample is brought into contact with the surface of a plate used for time-of-flight mass spectrometry, and the mass of a component captured on the plate surface is measured in time-of-flight type. The method of measuring with a mass spectrometer is mentioned.

The plate that can be adapted to the time-of-flight mass spectrometer may be any plate as long as it has a surface structure that can efficiently adsorb the peptide of the present invention to be detected. Such surface structures include, for example, functionalized glass, Si, Ge, GaAs, GaP, SiO, SiN, modified silicon, a wide range of gels or polymers (eg, (poly) tetrafluoroethylene.

Four

, (Poly) vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof). Examples of surface structures having a plurality of monomer or polymer sequences include linear and cyclic polymers of nucleic acids, polysaccharides, lipids, peptides having α-, / 3_ or ω_amino acids, and chromatography. Gel surface carriers (anionic / cationic compounds, hydrophobic compounds composed of carbon chains 1-18, hydrophilic compounds (eg, silica, nitrocellulose, cellulose acetate, agarose, etc.) cross-linked carriers), artificial Homopolymer (eg polyurethane, polyester, polycarbonate) (Bonate, Polyurea, Polyamide, Polyethyleneimine, Polyarylene sulfide, Polysiloxane, Polyimide, Polyacetate, etc.) and any of the above compounds are bound to known drugs or natural compounds (covalent and non-covalent bonds) Examples include coating with a heteropolymer.

[0017] In a preferred embodiment, the support used as the plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PV DF [usually As long as it is used in a plate for mass spectrometry, for example, insulator (glass, ceramics, plastic-resin, etc.), metal (aluminum, stainless steel, etc.), conductive polymer, etc. A force such as an aluminum plate is preferably used] (see WO 2 004/031759). The shape of the support is not limited to the force that can be appropriately devised into a shape suitable for the sample introduction port of the mass spectrometer to be used. As a plate for mass spectrometry coated with a thin layer of PVDF, preferably, Blotchip (registered trademark) manufactured by Protocera is used.

[0018] Preferably, the coating refers to a thin layer formed by being deposited on a support in a state where coating molecules are dispersed, rather than overlaying a preformed structure on the support, such as a membrane. . The manner in which the coating molecules are deposited is not particularly limited, but the means exemplified in the method for preparing a plate for mass spectrometry to be described later is preferably used, and the thickness of the thin layer depends on the transfer efficiency and mass of the molecules contained in the tissue or cells. A force that can be appropriately selected within a range that does not adversely affect the measurement sensitivity of analysis, for example

About 0.001 to about 100 m, preferably about 0.01 to about 30 m.

[0019] The mass analysis plate (support) can be prepared by a method known per se. For example, the preferred mass analysis plate described above has a thin coating on the support surface with a coating molecule such as PVDF. It is prepared by. Preferable examples of the coating means include coating, spraying, vapor deposition, dipping, printing (printing), and sputtering.

When “applying”, the coating molecules are applied in a suitable concentration (eg, about 1 to about 100 mg / mg of organic solvent such as dimethylformamide (DMF)). What is dissolved in about mL) (coating molecule-containing solution) can be applied to the substrate using an appropriate tool such as a brush.

In the case of “spraying”, the coating molecule-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the substrate.

When “evaporation” is performed, the coating molecule (which may be solid or solution) is heated and vaporized in a vacuum tank containing the substrate using a normal vacuum deposition apparatus for organic thin film production, and is then deposited on the surface of the substrate. A thin layer of the molecule can be formed.

In the case of “immersion”, the substrate may be immersed in a coating molecule-containing solution prepared in the same manner as described above.

In the case of “printing”, various printing techniques that can be normally used depending on the material of the base material can be appropriately selected and used. For example, screen printing is preferably used.

In the case of “sputtering”, for example, a DC high voltage is applied between the substrate and the coating molecule while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas collides with the molecule. The force S is applied to deposit the repelled coating molecules on the substrate to form a thin layer.

The coating may be applied to the entire surface of the substrate, or only to the surface (fraction) used for mass spectrometry.

[0020] The coating molecule can be used in a suitable form depending on the coating means, and can be applied to the substrate in the form of a coating molecule-containing solution, a coating molecule-containing vapor, a solid coating molecule, etc. It is preferable to apply in the form of a coating molecule-containing solution. “Apply” means to bring the coating molecule into contact with the support so that the coating molecules remain and deposit on the support after the contact. The amount of application is not particularly limited, but the coating molecular weight is, for example, about 10 to about 100,000 [I

Preferably, about 50 to about 5,000 m ² can be mentioned. After application, the solvent is removed by natural drying or vacuum drying.

[0021] The surface of the substrate in the mass spectrometry plate may be modified (processed) by an appropriate physical or chemical method in advance before coating with a coating molecule. Ingredients Specifically, techniques such as acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) for scratching the plate surface are exemplified.

[0022] In the transfer of the test sample to the mass spectrometry plate (support), the biological sample derived from the patient as the test sample is left untreated, or the high molecular protein is removed and concentrated by an antibody column or other method. Later, it is subjected to SDS-polyacrylamide gel electrophoresis or isoelectric focusing, and after electrophoresis the gel is brought into contact with the plate and transferred (plotted). A known transfer device can be used. The transfer method itself is known. Preferably electrotransfer is used. The sample developed on the gel after electrophoresis is transferred to the plate for mass spectrometry by various methods (diffusion, electric force, etc.). It is preferable to use a buffer having a pH of 7 to 9 and a low salt concentration for use in the electrotransfer. Specific examples include Tris buffer, phosphate buffer, borate buffer, and acetate buffer. Tris / Glycine / Methanol buffer, SDS-Tris / Tricine buffer, etc. for Tris buffer, ACN / NaCl / Isotonic phosphate buffer, Sodium phosphate / ACN, etc. Examples of the buffer solution include sodium borate-hydrochloric acid buffer solution, tris-borate / EDTA, and borate / ACN, and examples of the acetate buffer include tris-acetate / EDTA. Preferred are tris / glycine / methanol buffer and sodium borate monohydrochloride buffer. Examples of the composition of the tris / glycine / methanol buffer include Tris 10-15 mM, glycine 70-120 mM, and methanol 7-13%. Examples of the composition of the sodium borate monohydrochloride buffer include about 5 to 20 mM sodium borate.

[0023] Thereby, the molecules present in the test sample including the target molecules are efficiently captured on the support surface. After the plate is dried, a reagent called matrix is used to absorb the laser light and promote ionization of analyte molecules through energy transfer, which is advantageous for later mass spectrometry (when using MALDI method). Can also be added. As the matrix, those known in mass spectrometry can be used. For example, sinapimc acid (SPA ^ = 3, 5-dimethoxy-4-nydoroxycinammic acid)), nodole acrylate, IAA, 2,5-dihydroxybenzoic acid (2,5- force S including, but not limited to, dihydroxybenzoic acid (DHB), —cyan-4-hydroxycinnamic acid (CH CA), and the like. Preferably, it is DHB or CHCA [0024] The presence and amount of the peptide of the present invention, which is a target molecule, can be identified from information on the molecular weight by mass spectrometry of molecules in the test sample captured on the support surface by the above method. it can.

A mass spectrometer ionizes a gaseous sample, then puts the molecule or molecular fragment into an electromagnetic field, separates it by mass number / charge number from its movement state, and obtains the molecular weight of the substance, thereby obtaining the molecular weight of the substance. Is a device that measures and detects. A sample and a matrix that absorbs laser light are mixed, dried, crystallized, and ionized by energy transfer from the matrix and by instantaneous heating by laser irradiation, matrix-assisted laser deionization that leads the ionized analyte into the vacuum ( (MALDI) and time-of-flight mass spectrometry (TOFMS), which analyzes the number of masses by the time-of-flight difference of sample molecule ions by initial acceleration, and MALDI-TOFMS method. Direct electroionization, nanoelectrospray mass spectrometry (nano-ESMS), etc., in which sample solution is electrically sprayed into the atmosphere, and each analyte multivalent ion is led into the gas phase in an unfolded state A mass spectrometer based on the principle can be used.

A method for mass spectrometry of molecules on a plate for mass spectrometry is known per se. For example, the method described in W 0 2004/031759 can be appropriately modified as necessary.

[0025] From the result of mass spectrometry, based on the molecular weight information of the target molecule, the presence or absence and the amount of the target molecule in the test sample can be identified. In this step, it is also possible to output information from the mass spectrometer as differential information by comparing it with mass spectrometry data in a biological sample derived from a healthy person using an arbitrary program. . Such programs are well known, and those skilled in the art will be able to easily construct or modify such programs using known information processing techniques.

[0026] Particularly preferred! / In an embodiment! /, Each of the above steps was carried out using a plot tip of Protocera as a plate for mass spectrometry, and the MALDI type mass spectrometer was used for the present invention. Quantitative comparison (differential analysis) of peptides. Further, if necessary, the peptide remaining on the same chip can be identified. Alternatively, the test sample is quantitatively compared (differential analysis) using a protocera plot chip system, and the peptide It is also possible to carry out the identification using a combination of high performance liquid chromatography and an ion spray mass spectrometer (LC-MS / MS).

The measurement of the peptide of the present invention in the test method of the present invention can also be performed using an antibody against it. In this method, when an optimized immunoassay system is constructed and kitted, the peptide is detected with high sensitivity and high accuracy without using a special device such as the mass spectrometer. It is particularly useful in that it can.

[0028] The antibody against the peptide of the present invention can be prepared, for example, by isolating and purifying the peptide of the present invention from a biological sample derived from a patient expressing the peptide and immunizing an animal using the peptide as an antigen. it can. Alternatively, when the amount of peptide obtained is small, the peptide is partially digested with peptidase, etc., the amino acid sequence of the resulting fragment is determined by the Edman method, etc., and the peptide is encoded based on that sequence. The ability to obtain a cDNA encoding a protein containing the peptide by a hybridization method by synthesizing an oligonucleotide capable of hybridizing with a nucleic acid and using this as a probe in a cDNA library derived from the patient, or By performing RT-PCR using the oligonucleotide as a primer and RNA from the patient in a saddle shape, a cDNA fragment encoding the peptide is obtained, and the cDNA fragment is incorporated into an appropriate expression vector. A large amount of the peptide of the present invention is obtained by introducing it into a host cell and culturing the resulting transformant to collect the recombinant peptide. Can be prepared. Alternatively, the peptide of the present invention can also be obtained using the cell-free transcription / translation system using the cDNA obtained as described above in a saddle type. It can also be prepared in large quantities by organic synthesis.

[0029] As described above, the marker peptide of the present invention is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1. Therefore, an antibody against the peptide of the present invention can be synthesized, for example, by using a known peptide synthesis method based on the above amino acid sequence information, or it can be isolated or completely isolated from all or part of the amino acid sequence. It is desirable to cleave the fibrinogen A a chain with an appropriate peptidase or the like to obtain a peptide fragment containing all or part of the sequence of the peptide of the present invention, and prepare this as an immunogen.

[0030] The antibody against the marker peptide of the present invention (hereinafter sometimes referred to as "the antibody of the present invention") may be either a polyclonal antibody or a monoclonal antibody. It can be produced by epidemiological methods. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.

[0031] For example, the polyclonal antibody is a peptide of the present invention or a partial peptide thereof prepared by any one of the above methods or other methods (if necessary, urine serum albumin, KLH (Keyhole Limpet Hemocyanin) (It can also be a complex cross-linked to a carrier protein, etc.) as an antigen, with a commercially available adjuvant (for example, complete or incomplete Freund's adjuvant), and subcutaneously or intraperitoneally in animals every 2-3 weeks. Administer about 4 times (measure the antibody titer of the partially collected serum by a known antigen-antibody reaction and confirm its rise), collect whole blood about 3 to about 10 days after the final immunization, and It can be obtained by purifying serum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

[0032] In addition, monoclonal antibodies are produced by cell fusion methods (for example, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, "Monoclonal antibodies and basic cancer and clinical one", 2-14, Science Forum Publishing, 1985). For example, the peptide of the present invention or a partial peptide thereof is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and leukocytes are collected. Collect. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. Cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo, such as mice or rats, or preferably mouse ascites, and antibodies are obtained from the hybridoma culture supernatant and the ascites of the animals, respectively. be able to.

[0033] As the N-terminal fragment of fibrinogen A α chain, in addition to the peptide of the present invention, FPFP and fragments thereof other than the peptide of the present invention may also be present in the test sample. These peptides do not show significant variation in patients with presumptive cancer or CFS. Any of these can be significantly higher in disease. If the antibody to the peptide of the present invention is cross-reactive with one or more other N-terminal fragments of fibrinogen A α chain, the other disease is misdiagnosed as presumptive cancer or CFS (false positive) The possibility increases. Therefore, the antibody against the peptide of the present invention used in the test method of the present invention should be a highly specific antibody that does not cross-react with the fibrinogen Α α chain Ν terminal fragment other than the peptide. desirable. Such an antibody can be obtained by reacting a plurality of monoclonal antibodies obtained as described above with the other fragment and selecting an antibody that does not cross-react with them.

[0034] The test method of the present invention using the antibody of the present invention is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is chemically or physically limited. Any measurement method may be used as long as it is a measurement method which is detected by means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used.

Yes

[0035] Examples of the labeling agent used in the measurement method using a labeling substance include radioisotopes, enzymes, fluorescent substances, and luminescent substances. As the radioisotope, e.g., ^[12 ¾, ^[m I], ^[3 H], ^[14 C] and the like are used. As the above-mentioned enzymes, those which are stable and have a large specific activity are preferable. For example, / 3_galactosidase, / 3-darcosidase, alkaline phosphatase, baroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluoretcene isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can be used for binding an antibody or antigen to a labeling agent.

[0036] For insolubilization of an antigen or antibody, physical adsorption may be used, or a chemical bond that is usually used to insolubilize and immobilize proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass. [0037] In the sandwich method, the test sample is reacted with the insolubilized antibody of the present invention (primary reaction), and further labeled with another antibody of the present invention (secondary reaction), and then the insolubilized carrier By measuring the amount (activity) of the labeling agent, the amount of the peptide of the present invention in the test sample can be quantified. The primary and secondary reactions can be performed in the reverse order, or they can be performed at the same time! /.

[0038] The monoclonal antibody against the polypeptide of the present invention can also be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry and the like.

In the competition method, the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test sample is quantified. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is made of polyethylene glycol, a liquid phase method using a second antibody against the antibody, etc., and a force using a solid-phased antibody as the first antibody, Alternatively, the first antibody may be soluble and the second antibody may be a solid phase method using a solid phase antibody.

In the immunometric method, the antigen of the test sample and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test sample is excessively mixed with the antigen. After reacting an amount of the labeled antibody, the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to quantify the amount of antigen in the test sample.

In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate can be obtained.

[0039] In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations, and the like are not required to be set. The measurement system for the peptide of the present invention may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, refer to reviews, books, etc. And force S.

For example, Hiroshi Irie “Radio Imno Atssay” (Kodansha, published in 1974), Hiroshi Irie “Zone Radio Imno Atssey” (Kodansha, published in 1974), “Ezyme Immunoassay” edited by Eiji Ishikawa et al. "Medical Shoin, published in 1978", edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical School, published in 1982), edited by Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Methods in ENZYMOLOGY) Vol. 70 (Immunochemical Techniques (Part A)), Book Vol. 73 (Immunochemical Techniques (Part B), Id. Vol. 74 (Immunochemic al Techniques ( Part C '', Vol. 84 (Immunochemical Techniques (Part D: Selecte d Immunoassays), ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))) ibid Vol. 121 (Immunochemical Techniques (Part I: HybridomaTechnology and Monoclonal Antibodies)) Etc. can be referred to.

[0040] Alternatively, as another test method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer as described above, and the antibody on the probe is immobilized on the antibody. Examples include a method of contacting a test sample, subjecting a biological sample component captured by the antibody to mass spectrometry, and detecting a peak corresponding to the molecular weight of the marker peptide recognized by the antibody.

[0041] When the peptide of the present invention is detected with a statistically significant difference from the level in the body fluid of a healthy subject by any one of the above methods, It can be diagnosed that there is a high possibility of suffering from premature cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis or CFS definite stage.

[0042] The test method of the present invention is preferably performed by collecting a biological sample from a patient in time series and examining the temporal change in the expression of the peptide of the present invention in each sample. Sampling intervals of biological samples are not particularly limited, but do not impair the patient's QOL! / It is desirable to sample as frequently as possible in the heel range.For example, when using plasma or serum as a sample, about 1 minute to It is preferable to collect blood at intervals of about 12 hours. In the peptide of the present invention, the expression is remarkably increased at an early stage of CFS, and as the disease progresses, there is no significant difference from the level in the body fluid of healthy subjects. Therefore, when the level of the peptide of the present invention decreases with time, Determine the ability of the patient to improve the pathology of CFS, or the possibility that the pathology progresses further and shifts to the biodegradation syndrome, and is likely to be a shift! I can do it. In that case, the combination of other indicators of CFS diagnosis can be used to determine whether it is improving or worsening. Alternatively, the detection of various marker peptides for biodegradation syndrome previously discovered by the present inventors can monitor the transition of CFS patients to ecological collapse syndrome. Examples of such a marker peptide include a peptide consisting of 24 amino acids at the heel end of mature α 1 antitrypsin (α 1-AT), and an amino acid sequence 81 to 103 from the heel end of mature transthyretin (TTR). Peptide, mature apolipoprotein AI (ApoA-l) Peptide, mature apolipoprotein Β-ΙΟΟ (ΑροΒ-ΙΟΟ) each consisting of amino acids 62-83, 189-214 and 196-214 And a peptide comprising the amino acid sequence 3218 to 3249 from the N-terminus.

[0043] Further, the above-mentioned sampling method for spleen cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis or CFS is the same as the previous sampling. When a treatment for the disease is taken for the subject patient during the current sampling, it can be used to evaluate the therapeutic effect of the treatment. That is, for samples sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it is possible to evaluate that the effect of the treatment is effective. it can. On the other hand, if it is determined that the condition after treatment is not improved or worsened compared to the condition before treatment, it is evaluated that the effect of the treatment is effective. be able to.

[0044] Further, the peptide of the present invention can be used as a target for drug discovery for positive visceral cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis and CFS in addition to diagnosis. It can also be provided. That is, when the peptide itself has a physiological function in the direction of treatment (remission) of the disease (“therapeutic peptide”), by administering to the patient a substance that increases the amount or activity of the peptide, In addition, when the peptide itself has a physiological function in the direction of exacerbation of the disease (“exacerbation peptide” and! /, U), a substance that reduces the amount or activity of the peptide is administered to each of the diseases. Can be treated.

[0045] The present invention also provides a peptide of the present invention when the peptide of the present invention acts as a therapeutic peptide. By increasing the amount or activity of the peptide and / or by reducing the amount or activity of the peptide when the peptide of the present invention acts as an exacerbation peptide, Methods of treating ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis and / or CFS are provided. The therapeutic method specifically includes the effectiveness of a substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide. Including administering the amount to a patient with pancreatic cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis or CFS. Therefore, the present invention also comprises a substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide. A therapeutic agent for premature cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine fibroids, endometriosis or CFS is provided.

Specifically, examples of the substance that increases the activity of the peptide of the present invention as a therapeutic peptide include the peptide itself or a molecule having an agonist action similar thereto. Alternatively, as a substance that increases the activity of the peptide of the present invention as a therapeutic peptide, a non-neutralizing antibody, preferably an agonist antibody of the peptide can also be mentioned. On the other hand, examples of the substance that reduces the activity of the peptide of the present invention as an exacerbation peptide include a molecule having an antagonistic action of the peptide, or a neutralizing antibody against the peptide.

In addition, as a substance that increases the production of the peptide of the present invention as a therapeutic peptide, a degrading enzyme that releases the peptide from a protein (fibrinogen) present in the living body, the N-terminal side and the C-terminal side of the peptide Further comprising an amino acid sequence (eg, (Xaa) -Ala and Gly- (Xaa)) that is recognized and cleaved by the decomposing enzyme,

Examples include a substrate analog molecule, a molecule (including an analogous compound) that promotes the production of the decomposing enzyme, a molecule that promotes the activity of the decomposing enzyme, and a molecule that suppresses the production of an inhibitor of the decomposing enzyme. The amino acid sequence at the N-terminal side and C-terminal side of the peptide suggests the presence of a degrading enzyme that liberates the peptide, and the degrading enzyme using the amino acid sequence at the N-terminal side and / or C-terminal side of the peptide as a probe Search and identification are possible. Identified in this way A substrate or substrate analog molecule of a degrading enzyme, that is, a peptide molecule further comprising an amino acid sequence that is recognized and cleaved by the decomposing enzyme on the N-terminal side and C-terminal side of the peptide is decomposed in the body of a biodegradation syndrome patient. Since it is cleaved by the enzyme to release the peptide of the present invention or its analog molecule as a therapeutic peptide, it can exert the same therapeutic effect. On the other hand, substances that promote the production and / or activity of the identified degrading enzymes can also indirectly increase the production of the peptides of the invention as therapeutic peptides. These substances can be screened or molecularly designed by a method known per se once the target degrading enzyme is identified.

On the other hand, the substance that reduces the production of the peptide of the present invention as an exacerbation peptide includes a molecule that suppresses the production of a degrading enzyme that liberates the peptide from a protein present in the living body, an inhibitor of the decomposing enzyme, and an inhibitor of the inhibitor. Examples include molecules that promote production. The degrading enzyme that liberates the peptide of the present invention as an exacerbation peptide can be searched and identified by the same technique as that of the peptide of the present invention as the therapeutic peptide. Screening for substances that directly or indirectly suppress (inhibit) the production or activity of the decomposing enzyme using the thus-identified degrading enzyme by a method known per se, and a certain molecule is designed as a molecule. be able to.

A substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide can be formulated according to conventional means.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules). Syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as propellants. The force, the injection is according to a method known per se, for example, It is prepared by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily solution usually used for injections. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol) and nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration are prepared by mixing the above-mentioned compound or a salt thereof with a normal suppository base.

[0047] The oral or parenteral pharmaceutical composition described above is conveniently prepared in dosage unit form that is compatible with the dosage of the active ingredient. Examples of the dosage form of such a dosage unit are tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg per dosage unit form, especially 5 to 5 for injections. 100 mg, in other dosage forms 10-250 mg of the above compound is contained!

Each of the above-mentioned compositions is preferably mixed with a substance that increases the amount or activity of the peptide of the present invention as the therapeutic peptide or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide. Other active ingredients may be included as long as no interaction occurs.

[0048] Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, orally or parenterally to humans.

A substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide are determined by its action, administration route, patient severity, Although there are differences depending on age, body weight, drug acceptability, etc., for example, the amount of active ingredient per day for an adult is about 0.008 to about 25 mg / kg, preferably about 0.008 to about 2 mg / kg. Yes, this can be done in one or several divided doses.

[0049] The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples. Don't be! / Needless to say! / ...

Example

[0050] Example 1 Profiling analysis using BlotChip

After mixing 1.5 liters of serum from CFS and various cancer patients and healthy subjects with 4.5 L of sample treatment solution for electrophoresis (NuPAGE (registered trademark) LDS Sample Buffer 4x; Invitrogen) and heat-treated at 70 ° C for 10 minutes It was applied to 4-12% gradient polyacrylamide gel (Invitorigen) and electrophoresed. After completion of electrophoresis, the gel was cut out, laminated on BLOTCHIP (registered trademark) (Protosera, Inc.), and transferred to 90 mA for 120 minutes in an electrotransfer buffer (BLOTBuffer ™; Protosera, Inc.). After the transfer, rinse the surface of the chip with ultrapure water, apply matrix (a-Cyano-4-hydroxy cinamic acid) to the entire chip, and then matrix-assisted laser desorptionionization time-of- flight (MALDI- TOF) mass spectrometry was performed using a mass spectrometer (Ultra-FlexII manufactured by Bruker Daltnics). The measurement parameters were Detector voltage 1685V, Supression lOOO, Laser Intensity 28-35 Fuzzy mode, 4 15 points per chip, 500 times laser irradiation per chip, total 207,500 times laser irradiation. Each peak intensity in the obtained spectrum was integrated for each M / z and converted into one integrated spectrum. Differential profiling analysis was performed between healthy subjects' sera. Furthermore, the analysis results obtained in this way were collated with the peaks in the actual integrated spectrum.

As a result, a peptide with a molecular weight of about 1466 was detected in the CFS definite patient group, which was about 16 times as high as the peak strength group of healthy subjects (Table 1). The peptide is an effective early diagnostic marker for CFS because it is not significantly different from healthy individuals in CFS exacerbation, DIC and MOF stages (Figure 1).

[0051] [Table 1] Ratio of peptide peak to CFS positive sm and healthy subjects

¾¾

M / z CFS confirmation (A) Healthy subject (B) (A) / (B) P value

1465. 51 230, 312. 375 14, 227. 35 16. 2 3. 66197E-7 [0052] In addition to patients with CFS, the peptide may be used in patients with spleen cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, endometriosis, and uterine fibroids. (Figure 2 and Table 2). In particular, in presumptive cancer, the value was about 15 times higher than that of healthy subjects even if the histological type and staging were ignored.

[0053] [Table 2] Peptide peak ratio in each disease

The peak intensity of prostate cancer patients was L00.

[0054] Next, as a result of measuring and comparing the peak intensities of the peptides for each histological type in patients with presumptive cancer, they were positioned at stage 0, which is the earliest of all 6 stages of staging (1997, UICC). The peptide was clearly detected even in intraepithelial carcinoma (Tis) (Fig. 3 and Table 3). This means that even in stage II (2 cm or more, with metastasis), histopathological microinvasion can be diagnosed! / In other words, it cannot be diagnosed by imaging (MRI, CT, etc.). This means that early diagnosis of “invisible cancer” may be possible. [0055] [Table 3]

Ratio of peptide peak in spleen patient clinical stage

The peak of W-type unknown pancreatic cancer patients was set to 1.00 <

Example 2 Peptide identification by de novo MS / MS analysis on BlotChip

Using this (ma matnx— assisted laser desorption ionization time—of— flight (MALDI-TOF) mass spectrometer (Ultra-FlexII manufactured by Bruker Daltnics), Bradykinin, Angiote nsinll, Angiotensinl, SubstanceP, Bombesin, ReninSubstrate, ACTH Clip { l_17}, ACT H Clip {18-39}, mass calibrated using Somatostatin, then combined with NCBInr and SwissProt database through MASCOT search engine built into reflectron measurement iotools (Bruker Daltonik GmbH) As a result, the peptide was identified as a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1. As a result of homology search, the peptide was identified as It was revealed that FPA, a thrombin degradation product of fibrinogen, was a peptide further deleted from the N-terminal Ala residue.

Industrial applicability

[0057] The novel spleen cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterine leiomyoma, endometriosis and CFS diagnostic system of the present invention enable rapid and accurate diagnosis of the disease state. Therefore, it is useful in that it enables early detection and early treatment of the disease. In addition, since the peptide of the present invention, which is a measurement target in the present invention, can itself be a drug discovery target in these diseases, premature cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, uterus Screening for new therapies for fibroids, endometriosis and CFS, and It is extremely useful in that it can be used for treatment of the disease used.

While the present invention has been described with emphasis on the preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

This application is based on Japanese Patent Application No. 2006-335121 filed in Japan, the contents of which are hereby incorporated by reference. In addition, the contents described in all publications including the patents and patent application specifications mentioned here are incorporated herein by reference to the same extent as if all of them were explicitly stated. Things

Claims

The scope of the claims

[1] Measures the amount of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in a biological sample collected from the subject, wherein the subject has premature cancer, breast cancer, endometrial cancer, ovarian cancer, A test method for diagnosing a disease selected from the group consisting of cervical cancer, uterine fibroids, endometriosis, and Crow'Fukase syndrome definite stage.

[2] The method according to claim 1, wherein the biological sample is a body fluid.

[3] The body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid. The method described.

[4] The method according to any one of claims 1 to 3, comprising subjecting the biological sample to mass spectrometry.

[5] Use an antibody that specifically recognizes the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

A method according to any of claims 1 to 3, characterized in that V.

[6] A biological sample collected from a patient in time series, and the time course of the amount of peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sample is examined. One of the methods described.

[7] Evaluation of therapeutic effect in patients with diseases selected from the group consisting of premature stage of Fukase syndrome, premature cancer, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, uterine fibroids, endometriosis and crow A method comprising collecting a biological sample from the patient before and after treatment and examining the change in the amount of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sample. .