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WO2004087921A1 - Peptides antigeniques de cancer solide d'origine humaine, polynucleotides codant ces peptides et utilisation de ces derniers - Google Patents

Peptides antigeniques de cancer solide d'origine humaine, polynucleotides codant ces peptides et utilisation de ces derniers Download PDF

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Publication number
WO2004087921A1
WO2004087921A1 PCT/JP2004/004424 JP2004004424W WO2004087921A1 WO 2004087921 A1 WO2004087921 A1 WO 2004087921A1 JP 2004004424 W JP2004004424 W JP 2004004424W WO 2004087921 A1 WO2004087921 A1 WO 2004087921A1
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Prior art keywords
antibody
solid cancer
cancer
polynucleotide
peptide
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English (en)
Japanese (ja)
Inventor
Hideaki Shimada
Takenori Ochiai
Masaki Takiguchi
Takaki Hiwasa
Takeshi Tomonaga
Kazuyuki Matsushita
Fumio Nomura
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Japan Science and Technology Agency
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Japan Science and Technology Agency
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE

Definitions

  • the invention of this application relates to an antigen peptide of human solid cancer, a polynucleotide encoding the antigen peptide, and a method for diagnosing solid cancer using the same.
  • Solids such as esophageal cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, parotid gland cancer, head and neck cancer, bone and soft tissue sarcoma, ureteral cancer, bladder cancer, uterine cancer, liver cancer, breast cancer, ovarian cancer, fallopian tube, etc. All cancers are malignant tumors, especially advanced solid cancers that are difficult to treat and often fatal. Therefore, early detection of carcinoma is the most important issue as a countermeasure against solid cancer.
  • cancer tissue-specific serum markers such as ⁇ -phytoprotein, CEA, SCC, CA19-9, CYFR21-1, and the progress of treatment. Is being done. This method does not require large-scale equipment and places little burden on the subject, so it can be widely applied to many subjects without subjective symptoms.
  • all of these cancer markers have a positive rate of only about 20 to 30%, and most of them are negative especially in early stage cancer, and as a means for early detection of solid cancer. Was not enough.
  • methods for treating solid cancer include surgical removal of cancer tissue and systemic administration of anticancer drugs.
  • these treatments have little effect in the case of advanced solid tumors, and even if they are detected early, these treatments are extremely burdensome on the patient.
  • an antigen protein marker for example, Japanese Patent Application Laid-Open No. 7-51065, Japanese Unexamined Patent Application Publication No. No. 0-51 1536 is known.
  • the S EREX method which screens a protein made from mRNA of tumor cells of a cancer-bearing patient with the patient's autologous serum.
  • Japanese Patent Application Laid-Open No. 2001-333782 discloses a malignant melanoma antigen protein identified by the SEREX method, a DNA sequence encoding the same, and a method for diagnosing malignant melanoma using the same. . Disclosure of the invention
  • the invention of this application has been made in view of the circumstances described above, and has as its object to provide a novel antigenic peptide which is effective for diagnosis and treatment of solid cancer. Another object of the invention of this application is to provide a genetic material encoding the antigen peptide and an antibody against the antigen peptide.
  • an object of the invention of this application is to provide a method for diagnosing solid cancer and a method for treating solid cancer using the above-mentioned peptide, polynucleotide and antibody.
  • This application provides the following inventions (1) to (18) to solve the above problems.
  • (1) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 1, 13, 15, 17, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
  • a test is performed to determine whether or not a biological sample of a subject contains an antigen peptide that binds to each of the antibodies of the invention (4).
  • a method for diagnosing human solid cancer characterized by determining that the patient is a solid cancer patient or a solid cancer high-risk person.
  • the abundance of the polynucleotide of the invention (2) in a biological sample of a subject is tested, and a subject in which the abundance of one or more polynucleotides is higher than that of a healthy subject is referred to as a solid cancer.
  • a method for diagnosing human solid cancer characterized by determining that the patient is a patient or a person at high risk for solid cancer.
  • a diagnostic kit for solid cancer comprising:
  • a diagnostic kit for solid cancer comprising:
  • a solid cancer diagnostic kit comprising at least one kind of the antibody of the invention (4) and / or its labeled antibody.
  • a diagnostic kit for solid cancer comprising:
  • a diagnostic kit for solid cancer comprising:
  • a primer set for PCR-amplifying the polynucleotide of the invention (2).
  • a method for treating solid cancer comprising using the oligonucleotide or polynucleotide according to the invention (3).
  • Figure 1 shows Western blots showing the reactivity of the antigen peptide (SEQ ID NO: 22) expressed by the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 21 with the antibody in patient serum.
  • 4 is a photograph showing a result of a lot analysis.
  • FIG. 2 shows the results of Western blot analysis showing the reactivity between the antigen peptide (SEQ ID NO: 24) expressed by the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 23 and the antibody in the patient's serum. It is a photograph.
  • Figure 3 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 26) expressed by the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 25 with the antibody in the patient's serum. It is a photograph shown.
  • Figure 4 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 28) expressed by the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 27 with the antibody in the patient's serum. It is a photograph.
  • Figure 5 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 30) expressed by the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 29 with the antibody in the patient's serum. It is a photograph shown.
  • Figure 6 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 32) expressed by the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 31 with the antibody in the patient's serum. It is a photograph shown.
  • Figure 7 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 34) expressed by the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 33 with the antibody in the patient's serum. It is a photograph.
  • Figure 8 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 36) expressed by the polynucleotide shown in SEQ ID NO: 35 with the antibody in patient serum. It is a photograph.
  • Figure 9 shows the results of Western blot analysis showing the reactivity of the antigen peptide (SEQ ID NO: 38) expressed by the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 37 with the antibody in the patient's serum. It is a photograph shown.
  • FIG. 10 is a photograph showing the result of Western blot analysis showing the reactivity of the antigen peptide expressed by the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 39 with the antibody in the patient's serum.
  • FIG. 11 shows an antigen expressed by the polynucleotide having the nucleotide sequence of SEQ ID NO: 40.
  • 4 is a photograph showing the result of Western blot analysis showing the reactivity of a peptide with an antibody in a patient's serum.
  • FIG. 12 is a photograph showing the result of Western blot analysis showing the reactivity of the antigen peptide expressed by the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 41 with the antibody in the patient's serum.
  • the invention of this application is based on the novel antigenic peptide as described above, an antibody against the peptide, and a polynucleotide encoding them.
  • protein and “peptide” are composed of a plurality of amino acid residues linked to each other by amide bonds (peptide bonds). Means a molecule.
  • Polynucleotide is a phosphate ester of a nucleoside in which a purine or pyrimidine is linked to a sugar by a 3-N-glycosidic bond (ATP, GTP, CTP, UTP; or dATP, dGTP, dCTP ', dTTP)
  • oligonucleotide refers to a molecule consisting of 2 to 99 linked molecules.
  • one or more bases were added, deleted, substituted with other bases, or one or more amino acid residues were added or deleted based on these base mutations. And substitution to other amino acids.
  • serum antibody refers to an antibody IgG that is present in the serum of a solid cancer patient and binds to the antigenic peptide of the invention (1).
  • antibody of the invention (4) means a polyclonal antibody or a monoclonal antibody prepared using the antigen peptide of the invention (1) as an immunogen.
  • the antigen peptide of the invention (1) is a protein or peptide that expresses 22 kinds of genes or ESTs (Expressed Sequence Tags) shown in Table 1. Various functions are known for these gene products, but specific expression in solid tumors is not known. Also, the amino acid sequence of EST has not been specified, and its function is, of course, unknown.
  • the expression product of EST is defined as "a peptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 39-41" or "a peptide expressed by a genomic DNA hybridized with the polynucleotide".
  • SEQ ID NO: 120 was identified by two-dimensional electrophoresis, and the genes of SEQ ID NOs: 21-41 and EST were identified by SEREX method. is there.
  • the antigen peptide of the invention (1) can be used for the solid cancer diagnosis provided by the invention.
  • antigenic peptides can be expressed in vitro by, for example, preparing RNA by in vitro transcription from a recombinant expression vector having the polynucleotide of the invention (2) and performing in vitro translation using this as a type II.
  • a recombinant expression vector is introduced into prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells to produce transformed cells
  • peptides can be produced from the transformed cells. Can be expressed. .
  • the polynucleotide When expressing the antigen peptide by in vitro translation, the polynucleotide is introduced into a vector having an RNA polymerase motor to produce a recombinant expression vector, and this vector is used as a promoter.
  • the antigenic peptide can be produced in vitro if added to an in vitro translation system such as a egret reticulocyte lysate or a wheat germ extract containing the corresponding RNA polymerase.
  • examples of the RNA polymerase opening motor include T7, T3, SP6 and the like.
  • these vectors containing the NA polymerase promoter include pKA1, pCDM8, pT3 / ⁇ 718, ⁇ 7 / 319, pBluescriptII, and the like.
  • the polynucleotide When expressing an antigen peptide in a microorganism such as Escherichia coli, the polynucleotide is recombined into a vector having a replication origin, a promoter, a ribosome binding site, a DNA cloning site, and a terminator that can replicate in the microorganism. After preparing an expression vector, transforming a host cell with this expression vector, and culturing the resulting transformant, the microorganism can express the antigen peptide encoded by the polynucleotide. it can. At this time, a fusion protein with other proteins Can also be expressed. Examples of the expression vector for Escherichia coli include a PUC system, pBluescript II, a pET expression system, and a pGEX expression system.
  • the polynucleotide When the antigen peptide is expressed in eukaryotic cells, the polynucleotide is inserted into an eukaryotic cell expression vector having a promoter, a splicing region, and a poly (A) addition site to prepare a recombinant vector. When introduced into eukaryotic cells, the antigen peptide can be expressed in transformed eukaryotic cells.
  • expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3., PMSG, pYES2, etc. Can be illustrated.
  • pINDZV5-His When pINDZV5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP_C1, etc. are used as expression vectors, various tags such as His tag, FLAG tag, myc tag, HA tag, and GFP can be used.
  • An antigen peptide can also be expressed as an added fusion protein.
  • eukaryotic cells monkey kidney cells COS 7, cultured mammalian cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, African egg cells and the like are generally used. Any eukaryotic cell that can express the peptide may be used.
  • known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used.
  • the target peptide After expressing the antigen peptide in prokaryotic cells or eukaryotic cells, the target peptide can be isolated and purified from the culture by a combination of known separation procedures. For example, treatment with denaturing agents such as urea or surfactants, sonication, enzyme digestion, salting out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed phase chromatography.
  • the recombinant antigen peptide obtained by the above method includes a fusion protein with any other protein.
  • GST daltathione-S-transferase
  • GFP green fluorescent protein
  • peptides expressed in transformed cells are translated into individual cells within the cells. May undergo species modification. Therefore, modified peptides are also included in the scope of the antigenic peptides of the present invention. Examples of such post-translational modifications include N-terminal methionine elimination, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristylation, isoprenylation, phosphorylation and the like.
  • Invention (2) is a polynucleotide (DNA fragment; NA fragment) encoding the antigenic peptide of invention (1). Specifically, it is genomic DNA encoding each peptide (protein), mRNA transcribed from genomic DNA, and cDNA synthesized from mRNA. It may be double-stranded or single-stranded. Furthermore, the sense strand and antisense strand of these genomic DNAs, mRNAs and cDNAs are also included. Further, in the case of genomic DNA, the expression control region (promoter, enhansa, suppressor region) is also included.
  • cDNA can be synthesized and obtained by a method of isolating each cDNA using a probe DNA prepared based on the nucleotide sequence of SEQ ID NO: 1 or 3.
  • each cDNA can also be obtained by the RT-PCR method using the primer set provided by the present invention to convert mRNA isolated from human cells into type III.
  • the obtained cDNA is subjected to, for example, PCR (Polymerase Chain Reaction), NASBA (Nucleic acid seauence based amplification), TMA
  • invention (3) is an oligonucleotide or polynucleotide constituting the expression control region of the polynucleotide (particularly genomic DNA) of the invention (2).
  • Such an oligonucleotide, polynucleotide, or reotide can be obtained, for example, by a method of searching a known human genome database.
  • the oligonucleotide or polynucleotide of the present invention (3) can be used, for example, in the method for treating solid cancer of the present invention.
  • the antibody of the invention (4) is a polyclonal antibody or a monoclonal antibody.
  • the whole molecule which can bind to the epitope of the antigenic peptide of the invention (1), F ab, F (ab ') 2 , Fv fragment, etc. are all included.
  • a polyclonal antibody such an antibody can be obtained from serum after immunizing an animal using an antigen peptide or a partial fragment thereof as an immunogen.
  • it can be prepared by introducing the expression vector for eukaryotic cells into muscle or skin of an animal by injection or gene gun, and then collecting serum.
  • animals mice, rats, egrets, goats, and chickens are used.
  • Monoclonal antibodies can be prepared by a known monoclonal antibody preparation method (“monoclonal antibody”, written by Kamei Nagamune and Hiroshi Terada, Hirokawa Shoten, 1990; “Monoclonal Ant ibody” James W. Goding, third edition) , Academic Press, 1996).
  • the antibody of the present invention (4) also includes an antibody labeled with a labeling substance.
  • a labeling substance an enzyme, a radioisotope or a fluorescent dye can be used.
  • Enzymes are not particularly limited as long as they satisfy conditions such as a high turnover number, stability even when bound to an antibody, and specific coloring of the substrate. Enzymes to be used, for example, peroxidase, j8-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphoryl dehydrogenase, malate dehydrogenase and the like can also be used. In addition, enzyme inhibitors, coenzymes, and the like can also be used.
  • the binding between the enzyme and the antibody can be performed by a known method using a crosslinking agent such as a maleimide compound.
  • a crosslinking agent such as a maleimide compound.
  • a known substance can be used depending on the type of the enzyme to be used. For example, when using peroxidase as an enzyme, use 3,3 ', 5,5'-tetramethylbenzicin.When using alkaline phosphatase as an enzyme, use paranitrophenol or the like. Can be.
  • the radioactive isotope can be used those used in ordinary RIA, such as 1 2 5 I and 3 H.
  • the fluorescent dye those used in a usual fluorescent antibody method such as fluorescein isothiocyanate (FITC) -tetramethylrhodamine isothiocyanate (TRITC) can be used.
  • a metal such as manganese or iron is bound to the labeled antibody of the present invention. Things are also included. By administering such a metal-bound antibody to the body and measuring the metal by MRI or the like, the presence of cancer cells that produce the antigen-peptide bound to the antibody can be detected.
  • the method for diagnosing human solid cancer according to the invention (5) comprises testing whether or not one or more antibodies binding to each of the antigenic peptides according to the invention (1) are present in the serum of the subject.
  • a subject in which the antibody is present is determined as a solid cancer patient or a solid cancer high-risk subject. That is, since each of the antigen peptides of the invention (1) is a peptide that binds to an antibody (IgG) in the serum of a solid cancer patient, it is reacted with the serum of a subject to produce an antibody that binds to these antigen peptides. Can be determined as the serum of a solid cancer patient or a high-risk patient thereof.
  • the binding of the antigenic peptide to serum antibodies is determined for two or more, preferably five or more, more preferably ten or more, and most preferably 15 to 22 antigen peptides. Still further, other known solid cancer markers can be used in combination.
  • the serum of the subject is brought into contact with the antigen peptide, and the antigen peptide is reacted with the IgG antibody in the serum of the subject in a liquid phase.
  • a signal of the labeled IgG antibody may be detected by reacting the labeled IgG antibody that specifically binds to the IgG antibody in the serum.
  • an enzyme, a radioisotope or a fluorescent dye as exemplified in the above-mentioned labeled 'antibody can be used.
  • the enzyme activity is determined by optically measuring the amount of decomposition of the substrate, converted to the amount of bound antibody, and compared with a standard value. Is used to calculate the amount of antibody.
  • radioisotopes measure the radiation dose emitted by the radioisotope using a scintillation counter.
  • the amount of fluorescence may be measured by a measuring device combined with a fluorescent microscope.
  • the conjugate of antigen peptide + serum antibody + labeled IgG antibody is separated by a known separation method (chromatography, salting out, alcohol precipitation, enzyme method, solid phase method, etc.) and labeled.
  • a signal of a compounded IgG antibody may be detected.
  • the diagnostic kit of the invention (10) is provided.
  • the diagnostic method of the invention (5) is also carried out as a method (invention (6)) of immobilizing one or more antigen peptides on a plate or a membrane and testing the binding of the subject's serum to the antibody on the substrate. You can also. By immobilizing the antigen peptide on the substrate, unbound labeled binding molecules can be easily removed.
  • the diagnostic kit of the invention (11) is provided as one that enables simple and wide-ranging implementation of such a diagnostic method. In particular, in the protein array method using a membrane in which dozens of antigen peptides are immobilized, the expression of many types of antibodies can be analyzed in a short time using about 0.01 ml of the subject's serum. .
  • the diagnostic method of the invention (7) is to test whether or not the antibody of the invention (4) or an antigen peptide that binds to the labeled antibody is present in a biological sample of a subject,
  • the subject in which the antigen peptide is present is determined to be a solid cancer patient or a high-risk subject. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to an antigenic peptide expressed in colorectal cancer cells, a biological sample containing the antigenic peptide that binds to this antibody is used for solid cancer patients. Alternatively, it can be determined as a sample of the high-risk patient.
  • the binding of the antibody to the antigen peptide in the sample is preferably determined for at least two types, preferably at least five types, more preferably at least ten types, most preferably at least 15 types of antibodies.
  • blood and blood cells can be paired as a biological sample.
  • One embodiment of the diagnostic method of the present invention (7) is a method of binding an antibody and an antigen peptide in a liquid phase system.
  • the labeled antibody of the invention (4) is brought into contact with a biological sample to bind the labeled antibody to the antigen peptide, the conjugate is separated by the same method as in the above invention (5), and the labeling signal is similarly determined.
  • the diagnostic kit of the present invention (12) is provided as a method that enables such a diagnostic method to be carried out simply and widely.
  • Another method of diagnosis in a liquid phase system is as follows: the antibody (primary antibody) of the invention (4) is brought into contact with a biological sample to bind the primary antibody to the antigenic peptide; ) And detect the labeled signal in the conjugate of the three.
  • unlabeled secondary antibody is first The secondary antibody may be bound to a peptide conjugate and a labeling substance may be bound thereto. Such binding of the labeling substance to the secondary antibody can be carried out, for example, by biotinylating the secondary antibody and avidinizing the labeling substance.
  • an antibody a tertiary antibody recognizing a partial region (eg, Fc region) of the secondary antibody may be labeled, and the tertiary antibody may be bound to the secondary antibody.
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of a signal can be performed in the same manner as in the invention (5).
  • the diagnostic kit according to the invention (13) is provided as a method that enables simple and wide-ranging implementation of such a diagnostic method.
  • Another aspect of the diagnostic method of the invention (7) is a method of testing the binding between an antibody and an antigen peptide in a solid phase system.
  • This method using a solid phase system is a preferable method for detecting a trace amount of antigen peptide and simplifying the operation.
  • this solid phase method involves immobilizing the antibody (primary antibody) of the invention (4) on a resin plate or a membrane, binding the antigen peptide to the immobilized antibody, and washing and removing the unbound peptide.
  • a labeled antibody (secondary antibody) is bound to the antibody + antigen peptide conjugate remaining on the plate, and the signal of this secondary antibody is detected.
  • This method is a so-called “sandwich method”, and when an enzyme is used as a marker, it is widely used as “ELISA (enzyme-inked iinnmnosorbent assay)”.
  • ELISA enzyme-inked iinnmnosorbent assay
  • the primary antibody and the secondary antibody both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Detection of the signal can be performed in the same manner as in the invention (5).
  • the diagnostic kit of the invention (14) is provided as a method that enables such a diagnostic method to be carried out simply and widely.
  • the diagnostic kits of the inventions (10) to (14) are reagent kits for performing the diagnostic methods of the inventions (5) to (8). Various such kits are commercially available depending on the type of the test sample ⁇ .
  • the diagnostic kit of the present invention also uses the antigenic peptide, antibody and / or labeled antibody provided by the present invention. Except for this, it can be constituted by each element used in a publicly known kit.
  • the diagnostic method according to the invention (9) is a method for testing the abundance of each of the polynucleotides of the invention (2) in a biological sample of a subject, wherein the subject has a higher abundance of one or more polynucleotides than those of a healthy subject. Is determined to be a solid cancer patient or a person at high risk. As a specific criterion, the abundance of the polynucleotide of the subject is 10% or more, preferably 30% or more, more preferably 70% or more, and most preferably 100% or more compared to that of a healthy subject. Is
  • the biological sample can be stool, blood, or blood cells (eg, mononuclear cells).
  • the detection and measurement of the polynucleotide can be performed by a known PCR method, RT-PCR method, quantitative RT-PCT method, or the like, and in that case, the primer set of the invention (15) can be used for PCR.
  • primer sets have the sequence numbers 1, 3, 5, 7, 9, 11, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 , 40 and 41, and can be prepared through each step of synthesis and purification.
  • the following points can be pointed out as points to keep in mind when designing primers.
  • the size (number of bases) of the primer is 15 to 40 bases, preferably 15 to 30 bases in consideration of satisfying the specific annealing with the type I DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective. Avoid the complementary sequence between both primers so that one or two pairs of primers consisting of the sense strand (5 'end) and the antisense strand (3' end) do not anneal to each other.
  • the GC content should be about 50%, so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on the Tm (melting temperature), select primers with a Tm value of 55-65 ° C and close to each other to obtain highly specific PCR products. It is also necessary to take care to adjust the final concentration of the primer used in PCR to about 0.1 to about 1 M.
  • commercially available software for designing primers for example, O1igo TM (manufactured by National Bioscience Inc.
  • invention (16) is a method for treating solid cancer using the oligonucleotide or polynucleotide of invention (3). Since the oligonucleotide or polynucleotide of the invention (3) (hereinafter referred to as “promoter sequence”) is an expression control region of a gene that is specifically expressed in solid cancer cells, an anticancer substance is added to this promoter sequence. When a therapeutic gene is prepared by linking polynucleotides encoding the same and administered to the body by a method such as gene therapy, the therapeutic gene can be specifically expressed in cancer cells.
  • Examples of a polynucleotide encoding a substance having an anticancer effect or a precursor of a substance having an anticancer effect include, for example, p53, simple virus thymidine kinase, interleukin 1, 2, 1 1, 2, 1 1 7, 18 and 18, gene DNA encoding cytosine kinase, peracylphosphoribosyltransferase, etc., and cDNA thereof can be used.
  • This promoter sequence can also be used in a therapeutic method in which an adenovirus or a herpes virus is proliferated specifically for cancer cells to lyse the cancer cells. That is, for example, by inserting a promoter sequence in front of the E1A region of adenovirus, the adenovirus proliferates specifically only in cancer cells and causes the cancer cells to thaw.
  • Invention (17) is a method for treating solid cancer using the antibody of the invention (4). Since the antibody of the invention (4) binds to an antigen peptide that is specifically expressed in cancer cells, the antibody is bound to a known anticancer substance or anticancer agent and administered to the body of the patient. Substances and anticancer agents can act specifically on cancer cells. In addition, as long as the antibody has a killing effect on cancer cells, a therapeutic effect can be obtained even by administering the antibody alone.
  • Invention (18) relates to a method for treating solid cancer by controlling the expression level of the antigenic peptide of invention (1). That is, since the antigenic peptide of the invention (1) is specifically expressed in cancer cells, it is highly possible that its expression causes the canceration of the cells. Therefore, suppressing the expression of this antigenic peptide is expected to have a therapeutic effect on canceration and progression of cells. Specifically, this treatment is performed by the antisense therapy using an antisense DNA strand against the polynucleotide (particularly mRNA) of the invention (2) or the RNA interference (RNAi) method using double-stranded RNA. be able to.
  • Example 1 Identification of antigenic peptide by two-dimensional electrophoresis
  • frozen specimens were collected from each of the cancerous and non-cancerous tissues immediately after submission of colorectal cancer and stored at 180 ° C.
  • An appropriate amount of this frozen sample was pomogenized in 9.5 MUrea, 2% CHAP S, 1% DTT, protease inhibitor complete (Roche) solution, and then 100,000 g in an ultra-high speed centrifuge (Hitachi). The supernatant (protein solution) was extracted and the protein concentration was identified by absorbance.
  • each protein obtained from cancerous and non-cancerous tissues was separated by agarose isoelectric focusing in the first dimension and by 12% Tris / Glycine / SDS polyacrylamide gel electrophoresis in the second dimension. did.
  • the separated proteins were stained with Coomassie Brilliant Blue R250 to detect spots where the expression level was increased in cancerous tissues compared to non-cancerous tissues.
  • the gel is cut out from this spot, the protein contained in the gel is digested with trypsin (Roche), and the resulting peptide is recovered.
  • the peptide is collected and analyzed using an ion trap mass spectrometer (ThermoQuest LCQ DECA XP). The sequence was determined.
  • Example 2 Identification of antigenic peptide by SEREX method
  • RNA was isolated from these cultured cells by guanidinium thiosinate-monochlorochloroform extraction, and o1igo-dT (Oligotex-dT30 super, TAKARA) The mRNA was purified by performing poly (A) selection twice using ()), and cDNA library of each cell was constructed using the obtained mRNA (5.7 IL g).
  • Each phage vector of the cDNA library of each cancer cell prepared above was infected with Escherichia coli XL1-B1ue and plaque was formed on a NZY agarose plate. Expression of each infected E. coli was induced by 1 OmM IPTG treatment, and the peptide encoded by each cDNA was expressed. This peptide was transferred to a nitrocellulose membrane (NitroBind: Osmonics), and TBS [0.5% TBS containing 661120 (1 OmM Tris-HC1 501111 ⁇ &CI; pH 7.5) After removing the adsorbed pacteriophage by washing with [1], non-specific reaction was suppressed with TBS-Tween containing 1% albumin. This filter was reacted with esophageal cancer patient serum at room temperature for 2 hours each.
  • Serum was isolated from patients and stored at 80 ° C. Immediately before use, TBS containing 1% by weight of albumin—Tween (TBS containing 0.5% of polyoxyethylene sorbitan monolaurate— (Tween) solution which was finally diluted 500-fold with a solution was used. The diluted serum was mixed with the E. coli lysate at a ratio of 1: 5, left at 4 ° C for 8 hours, centrifuged at 15,000 rpm for 20 minutes, and the supernatant was recovered. Was. Untreated serum was diluted 2000-fold as necessary.
  • the serum was reacted with the ditrocellulose membrane on which the above-described expression peptide was plotted at room temperature for 10 to 20 hours to identify the peptide to which the antibody in the serum reacted. That is, the reaction was carried out using an alkaline phosphatase-labeled anti-human IgG-F (ab ') 2 goat antibody (Jachson) diluted 5000-fold as a secondary antibody, and reacted with nitroblue tetrazolium (Wako).
  • a labeling signal was detected by an enzyme color reaction using bromo-4-monochloro-3-indolyl phosphate (Wako), and a colony corresponding to a positive color reaction site was collected from the agarose plate, and the SM buffer ( 1 00 mM NaC 1, 10mM of MgSO 4 for, 5 Omm of T ris - HC 1; H 7. dissolved in 5). Repeat the secondary and tertiary screening in the same manner as above until color-colonizing positive colonies are unified.Screen phage clones that react with IgG in the serum of 20 patients and 347 positive clones was isolated. ⁇ [3] Identification of new antigens
  • the insert DNA was amplified from the obtained positive clones by PCR, and the obtained PCR product was sequenced using Big Dye DNA SeQuencing Kit (ABI) and ABI Prism (Perkin Elmer). .
  • the polynucleotide (cDNA) sequences encoding the amino acids of these 12 novel antigenic peptides are SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40 and It has 41 base sequences.
  • the nucleotide sequences of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37 correspond to SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34, 36, and 38, respectively.
  • FIGS. 11 and 12 show the results of Western blot analysis for examining the binding reaction between these 12 types of novel antigenic peptides and antibodies in the serum of each patient.
  • arrows indicate polypeptides that specifically reacted with antibodies in patient serum. It was detected in the E. coli extract treated with IPTG, but not detected in the untreated E. coli extract, indicating that the polypeptide was derived from the introduced cDNA.
  • the present invention provides two novel antigenic peptides useful as diagnostic markers for colorectal cancer, and a method for diagnosing colorectal cancer using them. This enables early and highly accurate diagnosis of colorectal cancer.

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Abstract

La présente invention se rapporte à un certain nombre de nouveaux peptides antigéniques s'avérant efficaces pour diagnostiquer un cancer solide, à des anticorps dirigés contre ces peptides antigéniques et à un procédé de diagnostic du cancer du colon au moyen desdits peptides.
PCT/JP2004/004424 2003-03-31 2004-03-29 Peptides antigeniques de cancer solide d'origine humaine, polynucleotides codant ces peptides et utilisation de ces derniers Ceased WO2004087921A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2007071947A1 (fr) 2005-12-19 2007-06-28 University Of Hull Essai de criblage du cancer

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JP4806772B2 (ja) * 2006-04-12 2011-11-02 国立大学法人 千葉大学 ｅＩＦ４Ｈ発現抑制による癌細胞の増殖阻害方法

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JPH0477498A (ja) * 1990-07-20 1992-03-11 Otsuka Pharmaceut Co Ltd ヒトプロテアソーム
US5843655A (en) * 1995-09-18 1998-12-01 Affymetrix, Inc. Methods for testing oligonucleotide arrays
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US5843655A (en) * 1995-09-18 1998-12-01 Affymetrix, Inc. Methods for testing oligonucleotide arrays
JP2002508652A (ja) * 1996-08-23 2002-03-19 ヒューマン ジノーム サイエンシーズ，インコーポレイテッド 新規なヒト増殖因子
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007071947A1 (fr) 2005-12-19 2007-06-28 University Of Hull Essai de criblage du cancer

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