JP2006008627A - Reagent for diagnosis of malignant tumor and diagnostic method for malignant tumor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein that is useful for detecting the whole malignant tumors, for example, cancer and the like, as a diagnostic reagent, and provide a diagnostic method for malignant tumor that is effective for diagnosis for judging that the specimen is a malignant tumor or not by examining this protein, thus the diagnostic method is applied to the primary screening of medical examination to enable the early diagnosis of malignant tumors and attain the early treatment. <P>SOLUTION: This invention provides a protein that specifically interacting with antiPCNA (proliferating cell nuclear antigen) polyclonal antibody and has a larger molecular weight than the PCNA on the SDS-PAGE or its modified body, further factors that specifically interact with the protein or its modifiers, and kits, compositions and methods using them. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the diagnosis of malignant tumors. More specifically, the present invention relates to a diagnostic reagent effective for early diagnosis of malignant tumors such as cancer by measuring a protein specifically expressed in the serum of a malignant tumor subject such as cancer. About diagnostic methods

  A specific antibody against a marker protein for malignant tumors such as cancer and sarcoma is clinically used for protein immobilization in cancer diagnosis and research areas. So far, production of mouse monoclonal antibodies against marker proteins expressed by various cancer cells has been reported. And the method of diagnosing cancer by measuring the protein of a cancer specific antigen as a cancer marker using this mouse monoclonal antibody is reported.

  A diagnostic chip capable of simultaneously detecting a large number of proteins and collectively diagnosing a plurality of cancers is being developed from SomaLogic (Colorado, USA) (Patent Document 1).

  However, in the technique described in Patent Document 1, it is necessary to use various specific antibodies or specific oligonucleotides, and diagnosis of a plurality of cancers is realized by accumulation of antibodies and the like. However, in order to diagnose all cancers, it is necessary to prepare a set of antibodies covering all cancers, but this is very difficult and complicated.

  Therefore, in the field concerned, the advent of factors / means capable of comprehensively diagnosing all cancers collectively is awaited.

A reagent based on a protein called human aspartyl (asparaginyl) β-hydroxylase (HAAH) reported by Panacea Pharmaceuticals (Maryland), USA, is an enzyme generated in the body of a major cancer subject. The purpose of this method is to detect cancer throughout the body early in a single blood test. However, since this protein is also expressed in normal or healthy subjects, its detection efficiency is limited.
US Pat. No. 6,544,776 International Publication No. 02/092782

  Mouse monoclonal antibody field for marker proteins expressed by various cancer cells, HAAH, which is a protein synthesized in large quantities in tumor cells, but effective against a wide range of cancers, even in normal cells There was a problem that the positive rate was not obtained to the extent that it was synthesized and cancer was accurately diagnosed, and the subject could misdiagnose negative even though it was cancerous. Above all, cancer markers are organ-specific and could not be diagnosed collectively as malignant tumors.

  In the case of the US SomaLogic diagnostic chip, it is necessary to measure a plurality of proteins, and it is more complicated to handle. In addition, although cancer diagnosis can be performed, the origin of the diagnosis is different from cancer such as sarcoma. Malignant tumors have disadvantages that cannot be measured.

  Reagents using the protein called “HAAH” reported by Panacea Pharmaceuticals in the United States as an index are mainly enzymes produced from cancer, but the enzyme is relatively easy to lose activity, and this is also the case with SomaLogic. Similarly, malignant tumors such as sarcomas have disadvantages that cannot be measured.

  Therefore, the present invention provides a protein useful for the detection of all malignant tumors such as cancer as a diagnostic reagent, and measuring this protein enables effective malignancy for diagnosing whether or not a subject is a malignant tumor. This is a tumor diagnosis method, which is applied to primary screening of health checkups, and is intended to be treated early by early diagnosis of malignant tumors.

  In the present invention, a protein that specifically interacts with an anti-PCNA polyclonal antibody and has a molecular weight larger than PCNA on SDS-PAGE is unexpectedly and universally expressed in a sample of a cancer specimen. The above problems have been solved by finding this.

  Accordingly, the present invention provides the following.

  In one aspect, the present invention provides a protein (herein also referred to as a PCNA-related antigen) having a molecular weight higher than that of PCNA on SDS-PAGE, which specifically interacts with an anti-PCNA polyclonal antibody. To do. The molecular weight of the protein is greater than 36000 daltons, less than 43500 daltons, more preferably less than 40000 daltons, and even more preferably less than 38000 daltons. Here, the PCNA may have the amino acid sequence shown in SEQ ID NO: 2.

  In another aspect, the present invention provides an agent that specifically interacts with an anti-PCNA polyclonal antibody that specifically interacts with a protein having a higher molecular weight on SDS-PAGE than PCNA. Here, the PCNA may have the amino acid sequence shown in SEQ ID NO: 2.

  In one embodiment, the factor is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof.

  In another embodiment, the factor is an antibody or derivative thereof.

  In another embodiment, the factor is used as a probe.

  In another embodiment, the agent is labeled or can be labeled.

  In another embodiment, the label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen-antibody reaction.

  In yet another embodiment, the agent of the invention does not interact with PCNA.

  In another embodiment, the agent of the invention is a monoclonal antibody.

  In another aspect, the present invention provides a hybridoma that produces the monoclonal antibody of the present invention.

  In another aspect, the present invention provides a composition for diagnosis of a malignant tumor, comprising a protein that specifically interacts with an anti-PCNA polyclonal antibody and that has a higher molecular weight on SDS-PAGE than PCNA. To do.

  In another aspect, the present invention provides a malignant agent comprising a factor that specifically interacts with a protein having a molecular weight on SDS-PAGE that is greater than PCNA, which specifically interacts with an anti-PCNA polyclonal antibody. A composition (for example, a cancer diagnostic agent) for tumor diagnosis is provided. The cancer diagnostic agent of the present invention can be used, for example, for diagnosis of breast cancer, stomach cancer, esophageal cancer, colon cancer, liver cancer, prostate cancer, bile duct cancer, osteosarcoma, malignant fibrous histiocytoma, etc. .

In another aspect, the present invention provides a kit for diagnosis of a malignant tumor in a subject,
(A) a means for detecting a protein that specifically interacts with an anti-PCNA polyclonal antibody and has a molecular weight larger than that of PCNA on SDS-PAGE;
Here, the appearance or increase of the expression of the protein provides a kit that indicates that the subject has or is likely to have a malignant tumor.

In another aspect, the present invention is a method for diagnosis of a malignant tumor in a subject comprising
(A) obtaining a sample from the subject;
(B) a step of mixing the sample with a factor that specifically interacts with an anti-PCNA polyclonal antibody and specifically interacts with a protein having a molecular weight on SDS-PAGE larger than that of PCNA;
(C) measuring the amount of interaction between the sample and the factor;
(D) determining whether the amount of interaction is greater than the amount of healthy individuals;
A method comprising:

  In a preferred embodiment of this method, the malignant tumor comprises bile duct cancer, melanoma, lung cancer, stomach cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymic cancer, lymphoma, sarcoma, liver. Non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer (chest cancer), prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer (colorectal cancer), multiple myeloma, nerve Selected from the group consisting of blastoma, bladder cancer, cervical cancer, skin cancer, breast cancer, esophageal cancer, nephroma, brain tumor, osteosarcoma, Ewing sarcoma and malignant fibrohistiocytoma, More preferably, it is selected from the group consisting of bile duct cancer, prostate cancer, stomach cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, osteosarcoma, Ewing sarcoma and malignant fibrohistiocytoma Contains at least one tumor.

  In a more preferred embodiment, the malignant tumor is bile duct cancer, prostate cancer, stomach cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, osteosarcoma, Ewing sarcoma and malignant fibrohistiocytoma. All tumors selected from the group consisting of:

  In the present invention, it is possible to use a method in which the serum of a healthy person and the serum of a subject are used as serum, the target protein in the subject serum is measured, and the results are compared.

  In the present invention, this protein is not contained in the serum of a healthy person. Therefore, this protein is not detected when healthy human serum is used. However, this protein is expressed at a high rate in the serum of malignant tumor subjects such as cancer. Therefore, it can be diagnosed that the subject may be a malignant tumor such as cancer, based on the expression of the present protein observed using the serum of the subject.

The protein of the present invention proliferates remarkably in the serum of a malignant tumor subject such as cancer and is used as a tumor marker in malignant tumor diagnosis because its appearance frequency is specific to cancer and the like In fact, when diagnosing which organ is a malignant tumor, it can be diagnosed in combination with other known organ-specific cancer diagnostic methods and taking into account the results. preferable.

  The cancer diagnostic agent of the present invention can also be used as a marker for examining the presence or absence of cancer metastasis after surgery.

  Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  The present invention provides a comprehensive cancer marker that can diagnose any cancer or sarcoma regardless of the site. By using such a comprehensive marker, it becomes possible to diagnose cancer simply and easily.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it should be understood that the singular article also includes the plural concept thereof, unless otherwise specified. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, for example, Sambrook J. et al. (1989). Molecular Cloning. : A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) .Current Protocolsin Molecular Biology, Greene Pub. Associat ES and Wiley-Interscience; Ausubel, FM (1989). A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience; Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992) .Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, FM (1995) .Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Innis, MA et al (1995) PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, a separate volume of experimental medicine, “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, etc., and related parts (which may be all) are incorporated herein by reference. The

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) .Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, RL et al. (1992) .The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al (1994). AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc. Which are incorporated herein by reference in the relevant part.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

(Biochemistry)
As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to an amino acid polymer of any length. This polymer may be linear, branched or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. As used herein, the term is preferably linear and is composed of only natural amino acids, but is not limited to it, preferably in a form translated by a nucleic acid molecule. The term can also encompass one assembled into a complex of multiple polypeptide chains. This term also encompasses naturally occurring or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation (glycosylation), lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is included. The gene product of the present invention usually takes the form of a polypeptide. As used herein, the polypeptides of the present invention typically have a specific sequence or a variant thereof. Gene products of genes such as antigens (eg, antigens derived from cancer cells) usually take the form of polypeptides having such sequences. Here, sequences having modifications can also be used in the present invention for diagnostic, prophylactic and therapeutic purposes.

  In the present specification, the “sugar chain” refers to a compound formed by connecting one or more unit sugars (monosaccharide and / or a derivative thereof). When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond. Examples of such sugar chains include polysaccharides (glucose, galactose, mannose, fucose, xylos, N-acetylglucosamine, N-acetylgalactosamine, sial contained in the living body. Acid and their complexes and derivatives), as well as a wide range of sugar chains that are degraded or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, etc. It is not limited to them. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “carbohydrate”, and “carbohydrate”.

The "monosaccharide" in the present specification, not hydrolyzed in simpler molecules which refers to a compound represented by the general formula C _n H _2n O _n. Here, those in which n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are converted into diose, triose, tetrose, pentose, hexose, heptose, respectively. Socto, Octose, Nonose and Decose. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol, and the former is called an aldose and the latter is called a ketose.

  In the present specification, the “monosaccharide derivative” refers to a substance in which one or more hydroxyl groups on the monosaccharide are substituted with another substituent and the resulting substance is not within the range of the monosaccharide. Examples of such monosaccharide derivatives include sugars having a carboxyl group (for example, aldonic acids in which the C-1 position is oxidized to become carboxylic acids (for example, D-gluconic acid in which D-glucose is oxidized)). , Uronic acid in which terminal C atom is carboxylic acid (D-glucuronic acid in which D-glucose is oxidized), sugar having amino group or amino group derivative (for example, acetylated amino group) ( For example, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, etc., sugars having both amino group and carboxyl group (for example, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid Etc.), deoxylated sugars (for example, 2-deoxy-D-ribose), sulfated sugars containing sulfate groups, phosphorylated sugars containing phosphate groups, and the like. . Non Alternatively, Hemiaseta - in sugar forming the Le structure, alcohol - Le react with acetals - glycosides Le structure are also within the scope of the monosaccharide derivatives.

  As used herein, “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to a nucleotide polymer of any length. Examples of such nucleic acid molecules include, but are not limited to, cDNA, mRNA, and genomic DNA. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. As used herein, “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or has an unusual linkage between nucleotides, and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphate in an oligonucleotide. Derivative oligonucleotide in which diester bond is converted to N3′-P5 ′ phosphoramidate bond, derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide Oligonucleotide substituted with C-5 propynyluracil, derivative oligonucleotide substituted with C-5 thiazo-ruuracil in uracil in oligonucleotide, cytosine in oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotide, derivative oligonucleotide in which cytosine in oligonucleotide is substituted with phenoxazine-modified cytosine, and ribose in DNA is substituted with 2'-O-propylribose Examples include derivative oligonucleotides and derivative oligonucleotides in which the ribose in the oligonucleotide is substituted with 2'-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence is also a conservatively modified variant thereof (eg, a degenerate codon substitute), similar to a sequence explicitly shown in the sequence listing. , Complementary sequences, and corresponding sequences (eg, mouse sequences for human sequences, etc.) are contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzeret al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). Since the genetic vaccine of the present invention usually takes this polynucleotide form, and usually needs to be translated into an amino acid form, a form that is transcribed and translated in vivo (for example, consisting of natural nucleotides) Although it is preferable to take, it is not limited to it. As used herein, nucleic acids and nucleic acid molecules can be included within the concept of the term “gene”. Nucleic acid molecules that code for a gene sequence also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced so that different (another) nucleic acid splice products code for different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition. Thus, the gene of the present invention can also include splice variants thereof. Such mutants are useful in the diagnosis of the present invention.

  As used herein, “molecular weight” is used in the same meaning as commonly used in the art, and is expressed in daltons. In this specification, the molecular weight of a protein is typically expressed by the apparent molecular weight on SDS-PAGE. “Molecular weight is affected not only by the amino acid sequence but also by modification (for example, sugar chain etc.) on SDS-PAGE. Therefore, when the protein of the present invention is larger than PCNA, for example. In addition to differences in amino acid sequence, it includes that the molecular weight is different as a result of post-translational modification (eg, sugar chain, lipidation, etc.) Those skilled in the art have different molecular weights on SDS-PAGE. It is understood that this is easily confirmed by experimenting with the standard PCNA and the SDS-PAGE conditions described herein.

  Here, the conditions of SDS-PAGE used in this specification are as follows. SDS-PAGE uses a 12.5% gel. The gel is electrophoresed at 100 V for 1 hour and then transferred to Immobilon-P Transfer membrane (Millipore, Billerica, Mass., USA). Then, positive / negative is judged by Western blotting.

  As used herein, “gene” refers to a factor that defines a genetic trait. Those that define the primary structure of a protein are called structural genes, and those that influence the expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. Therefore, a gene for a protein usually includes both a structural gene of the protein and a transcriptional or translational regulatory sequence such as a promoter of the protein. As used herein, “gene” usually refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule”, but in some cases “protein”, “polypeptide”, “oligopeptide” and “peptide” Is understood. Those skilled in the art understand what “gene” refers to depending on its context. Also herein, “gene product” refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide” expressed by a gene. Is included. A person skilled in the art can understand what a gene product is, depending on the situation.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity indicate the same numerical value.

  In the present specification, the “amino acid” may be natural or non-natural as long as it satisfies the object of the present invention. As used herein, “derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.

  In the present specification, the “nucleotide” may be natural or non-natural. As used herein, “derivative nucleotide” or “nucleotide analog” refers to a nucleotide having a function different from that of a naturally occurring nucleotide but similar to that of the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). However, it is not limited to these. In the present invention, any analog may be used as long as the gene product is expressed. Preferably, a genetic vaccine containing natural nucleotides is used. This is because the natural type is highly likely to be translated into a peptide.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. It is understood that such a fragment can also be used for cell surface markers, cancer antigens and the like used in the present invention as long as they have the function (for example, antibody-inducing ability).

  As used herein, “corresponding” amino acids and nucleic acids (eg, in a cell surface marker) are the same as the predetermined amino acids and nucleic acids in a reference polypeptide and nucleic acid molecule, respectively, in a given polypeptide and nucleic acid molecule. For example, in an enzyme, an amino acid and a nucleic acid that are present at the same position as the active center of the enzyme and have a similar contribution to the catalytic activity Refers to the nucleic acid to be stored. If it is an antigen, it may correspond to an amino acid sequence relating to a part corresponding to an amino acid sequence involved in antigenicity (for example, epitopes), and if it is a nucleic acid sequence, the nucleic acid sequence or a specific part that it codes It can be a part that performs the same function as.

  As used herein, a “corresponding” gene (for example, a polypeptide such as a tumor marker, a nucleic acid molecule, etc.) has or has the same action as a predetermined gene in a species as a reference for comparison in a certain species. Is a gene that has the same origin in terms of evolution, when there are multiple genes having such an action. Thus, the corresponding gene of a gene can be an ortholog of that gene. Therefore, genes corresponding to genes such as human tumor markers can also be found in other animals (mouse, rat, pig, cow, etc.). Such corresponding genes can be identified using techniques well known in the art. Therefore, for example, a corresponding gene in an animal is obtained by using a sequence of a gene (for example, a gene such as a cancer antigen) serving as a reference for the corresponding gene as a query sequence. It can be found by searching the table or by screening the library in a wet experiment.

  As used herein, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. Electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448). (1988)), Smith and Waterman method (Smithand Waterman, J. Mol. Biol. 147: 195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 ( 1970)), and the like, but are not limited thereto. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes or microarrays (microarray assays) affixed to glass plates, PCR and in situ hybridization, etc. But not limited to. In the present specification, it is intended that cancer antigens and the like should also include corresponding genes identified by such electronic and biological searches.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by commonly recognized single letter codes.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using FASTA, which is a tool for sequence analysis.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such variants include one or several substitutions, additions and / or deletions, or one or more substitutions, additions and / or deletions relative to a reference nucleic acid molecule or polypeptide. But are not limited thereto. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the family of hemoglobin genes with multiple gene structures as an example, human and mouse cancer markers can be orthologs, but human cancer markers are also paralogs (genes generated by gene duplication) . Similarly, orthologs may exist for protozoan genes such as malaria. Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs can usually perform the same function as the original species in another species, the orthologs of the present invention can also be useful in the present invention. It is understood that orthologs are also present in the cell surface markers used in the present invention.

  In the present specification, “substitution”, “addition” and “deletion” of a polypeptide or polynucleotide refer to an amino acid or an alternative thereof, or a nucleotide or an alternative thereof, respectively, with respect to the original polypeptide or polynucleotide. Things that are replaced, added, and removed. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. There may be any number of substitutions, additions or deletions, as long as it is one or more, and such numbers may be used in variants having such substitutions, additions or deletions (eg, to proteasomes). As long as transport etc. is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length. It is understood that variants such as such tumor markers can be used in the present invention.

  In the present specification, “expression” of a gene product such as a gene, polynucleotide, or polypeptide means that the gene (usually in a DNA form) or the like undergoes a certain action in vivo to become another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form, but transcription to produce mRNA can also be a form of expression. In another embodiment, such a polypeptide form may have undergone post-translational processing (eg, leader sequence excision).

  In the present specification, the expression "specifically expresses" a gene means that the gene is expressed at a different level (preferably higher) at a specific site or time of an organism than other sites or times. . “Specific expression” may be expressed only at a certain site (for example, a specific site such as a diseased site) or may be expressed at other sites. The expression specifically preferably means that it is expressed only at a certain site. Such specific expression can be realized by utilizing the characteristics of antigen-presenting cells.

  As used herein, “detection” or “quantification” of gene expression (eg, mRNA expression, polypeptide expression) can be accomplished using appropriate methods, including, for example, mRNA measurement and immunoassay methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include ELISA method, RIA method, fluorescent antibody method, Western blot method, immunohistochemical staining method, etc., using a microtiter plate as necessary. . Examples of the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array). The DNA array is widely outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method"). The protein array is described in detail in Nat Genet. 2002 Dec; 32 Suppl: 526-32. Examples of gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like. Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.

  In the present specification, the “expression level” refers to an amount at which a polypeptide or mRNA is expressed in a target cell or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA method, RIA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method using the antibody of the present invention. The expression level of the polypeptide of the present invention at the protein level or the polypeptide of the present invention evaluated by any appropriate method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. The expression level at the mRNA level can be mentioned. “Change in expression level” means an increase in the expression level at the protein level or mRNA level of the polypeptide of the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Or it means to decrease.

  The present invention can be used on samples derived from various tissues or organs.

  As used herein, “tissue” refers to a population of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” usually has the same origin, but even cell populations with different origins can be referred to as a tissue if they have the same function and / or morphology. Such a tissue may be the subject of the diagnosis of the present invention. In nature, cell populations having two or more different origins may constitute a tissue. Even in such a case, the present invention can make such a tissue a diagnostic object. Usually, tissue constitutes part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis.

  In the present invention, for example, when an organ is targeted for diagnosis or the like, such an organ may be any organ, and the tissue or cell targeted by the present invention is derived from any organ or organ of an organism. It may be a thing. In the present specification, the term “organ” or “organ” is used interchangeably, and a function of an organism individual is localized and operated in a specific part of the individual, and that part has morphological independence. Is a structure. In general, in a multicellular organism (eg, an animal), an organ consists of several tissues with a specific spatial arrangement, and a tissue consists of many cells. Such organs or organs include organs or organs associated with the vascular system. In one embodiment, organs targeted by the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, brain, peripheral limbs, retina and the like. It is not limited to them.

(Antigens and antibodies)
In the present specification, the “antibody” refers to a protein produced in vivo by stimulation of an antigen in an immune reaction, or a variant thereof that specifically binds to the antigen. Such antibodies include, for example, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof such as F (ab ′ ) ₂ and Fab fragments, as well as other recombinantly produced conjugates. Furthermore, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, α-galactosidase, and the like. Such conjugates or fusions are therefore within the scope of antibody derivatives.

  As used herein, “monoclonal antibody” refers to an antibody that is substantially directed against only one antigenic determinant. Usually, such monoclonal antibodies can be made in pure form in vitro by hybridomas made by fusing antibody-producing B cells and myeloma cells.

  As used herein, “polyclonal antibody” refers to an antibody in which antibodies against a plurality of antigenic determinants of an antigen molecule are mixed. Examples include, but are not limited to, antiserum raised against a certain antigen, or an antiserum purified from a gamma globulin fraction, and those normally treated as antibodies.

  As used herein, “hybridoma” refers to a hybrid cell having tumorigenicity produced by artificially fusing two types of cells. When normally used herein, it refers to a hybrid cell of a plasmacytoma and a B cell (B lymphocyte). B cells isolated from immunized individuals can continue to make antibodies in culture forever by fusing with plasmacytoma cells.

  As used herein, “antigen” refers to any substrate that can be specifically bound by an antibody molecule. As used herein, “immunogen” refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response.

  As used herein, “epitop” refers to a group constituting a structure that determines an antigen. Thus, epitopes depend on a set of amino acid residues involved in recognition by a particular immunoglobulin, or in the case of T cells, by T cell receptor proteins and / or major histocompatibility complex (MHC) receptors. A set of amino acid residues necessary for recognition is included. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. In the present invention, there is no need to determine the epitopes. In the immune system field, in vivo or in vitro, an epitope is a characteristic of a molecule (eg, primary peptide structure, secondary peptide structure or tertiary peptide structure and charge), and is expressed by an immunoglobulin, T cell receptor or HLA molecule. Form a recognized site. Epitopes containing peptides may contain more than two amino acids in a spatial conformation unique to the epitopes. In general, an epitope consists of at least 5 such amino acids and typically consists of at least 6, 7, 8, 9, or 10 such amino acids. The length of the epitope is generally preferred because it is more similar to the antigenicity of the original peptide, but this may not always be the case considering the conformation. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance spectroscopy. Furthermore, the identification of epitopes in a given protein is readily accomplished using techniques well known in the art. For example, Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998 (a general method for the rapid synthesis of peptides to determine the location of immunogenic epitopes in a given antigen); No. 4,708,871 (Procedure for identifying and chemically synthesizing antigen epitopes); and Geysen et al. (1986) Molecular Immunology 23: 709 (high affinity for a given antibody). See Techniques for Identifying Peptides Having. Antibodies that recognize the same epitope can be identified in a simple immunoassay. Thus, methods for determining epitopes containing peptides are well known in the art, and such epitopes are well known to those skilled in the art once a primary sequence of nucleic acids or amino acids is provided. It can be determined using conventional techniques. Therefore, in the present invention, the epitopes of the protein of the present invention capable of distinguishing PCNA (that is, epitopes that do not react with PCNA but react with the protein of the present invention) and the like are used for such epitopic mapping. Etc. can be determined.

  Thus, for use as an epitope containing peptides, a sequence of at least 3 amino acids in length is required, preferably this sequence is at least 4 amino acids, more preferably 5 amino acids, 6 amino acids, 7 amino acids. , 8 amino acids, 9 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids in length may be required.

  As used herein, “probe” refers to a substance to be searched for in biological experiments such as screening in vitro and / or in vivo, such as a nucleic acid molecule containing a specific base sequence or Examples thereof include, but are not limited to, a peptide containing a specific amino acid sequence.

  Examples of proteins that are usually used as probes include, but are not limited to, antibodies or derivatives thereof. When a protein is used as the probe, it can be directly or indirectly labeled with a label.

  In the present specification, the “label” refers to a substance or factor added to know the behavior of an element or substance. Such labels include radioactive materials, chemiluminescent materials, fluorescent materials, phosphorescent materials, color formers, enzyme reaction substrates or enzymes, additional antibodies or antigens, ligands, haptens and their raw materials (ie, radioactive, chemical by conversion processes) A material exhibiting luminescence, fluorescence, phosphorescence, color development, etc.), but is not limited thereto.

(disease)
As used herein, “tumor” refers to an aggregate of cells exhibiting autonomous hyperproliferation, and includes malignant and benign ones. Tumors can include epithelial tumors (malignant are called cancer), non-epithelial tumors (malignant are called sarcomas), mixed tumors, teratomas, and the like. Benign include epithelial tumors such as nematodes and squamous papillomas; non-epithelial tumors such as fibromas and lipomas, mixed tumors such as pleomorphic nematodes, and teratomas such as mature teratomas Can do.

  In the present specification, the term “malignant tumor” is strongly atypical, has a rapid growth, invades the surrounding tissue destructively, causes metastasis, and causes the host to die, such as cancer, sarcoma, Includes mixed tumors, teratomas.

  As used herein, “tumor marker” refers to any factor (eg, protein, nucleic acid, other gene product, etc.) produced by a tumor. Preferably, those that do not show production in adult tissues are used. Conventional tumor markers have high specificity for specific cells (AFP, NSE, HCG, PSA, PAP, seminoprotein, hormone, MAM6), and are produced at specific times of cell differentiation (eg, leukemia differentiation) Antigens, SSEA1, PGP), and those with high specificity in pathogenesis (ATL antibody, EB antibody, HPV) and the like were known only. Factors that determine the blood concentration of the marker include, for example, production (number of marker-producing tumor cells / benign cells and activity); direction of secretion (eg local blood flow and lymph flow, glandular structure) Disorder, blocking excretion pathways, storage pools, etc.); metabolism and excretion (degrading organ function, metabolic disorders, circulatory failure, renal failure, binding proteins, presence of antibodies, etc.) It is not limited to. Tumor markers include high-risk screening, follow-up, adjunct diagnosis of cancer, differentiation of primary organ and cell type, estimation of stage and metastasis status and prognosis, assessment of therapeutic effect, monitoring of recurrence, cancer It can be used for grasping the biological characteristics of the drug and selecting a treatment method.

In particular, in the present specification, the “cancer antigen” refers to an antigen molecule that is newly expressed as normal cells become cancerous. Such cancer antigens include, but are not limited to, for example:
(1) Cancer virus-derived antigens (for example, T antigens derived from DNA-type tumor viruses such as adenovirus, poliovirus, SV40). In human and mouse RNA-type tumor viruses, viral envelope proteins are expressed on the cell surface.

(2) Tumor specific transplantation antigen (TSTA); this antigen is the target when cancer cells are rejected as a result of the establishment of a specific immune response against syngeneic cancer cells. Applicable are antigens. When a mutant protein is produced in a cancer cell due to a gene mutation, it is associated with a major histocompatibility gene complex (MHC) molecule in the cell as a peptide fragment, just like other normal intracellular proteins. It becomes expressed on the cell surface. ;
(3) Cancer associated antigen (TAA). An antigen that is not necessarily specific for cancer cells but shows characteristic expression with canceration. For example, α-fetoprotein in liver cancer, fetal cancer antigen (carcinoembryonicantigen, CEA) in intestinal cancer, and the like are applicable. These are proteins that originally exist only in normal fetuses and are not found in adult tissues. However, these proteins are called carcinoembryonic antigen (oncofetalantigen) because they re-express with canceration.

  As used herein, the cancer antigen can be used in any form, but in particular, a form called a cancer-associated antigen is preferably used. This is because it is expressed on the surface of cancer cells by association with MHC.

  In the present specification, “cancer” or “cancer” is used interchangeably, and is a malignant tumor or its malignant tumor that has strong atypia, can grow faster than normal cells, and can invade surrounding tissues destructively or metastasize. This refers to a state in which such a malignant tumor exists. In the present invention, cancer includes, but is not limited to, solid cancer and hematopoietic tumor. Examples of cancer include adenocarcinoma, cystadenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, malignant melanoma, choriocarcinoma, seminomas -Mammals, fetal cancer, etc., but are not limited thereto.

  In the present specification, “solid cancer” refers to cancer having a solid shape, and is a concept confronting hematopoietic tumors such as leukemia. Examples of such solid cancers include breast cancer, liver cancer, stomach cancer, lung cancer, head and neck cancer, cervical cancer, prostate cancer, retinoblastoma, malignant lymphoma, esophageal cancer, brain tumor, Examples include, but are not limited to, bone tumors. Hematopoietic tumors include, but are not limited to, leukemia.

  As used herein, “sarcoma” refers to a malignant tumor that arises from a non-epithelial tissue (usually excluding bone marrow lymphoid tissue) that is a supporting tissue of a living body. For example, fibrosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, lymphangiosarcoma, synovial sarcoma, malignant mesothelioma, invasive meningiomas, leukemia And malignant lymphoma.

  Examples of other malignant tumors include malignant mixed tumors, mixed tumors such as Wilmsoma, and teratomas such as immature teratomas.

  In the present specification, “cancer cell” refers to a cell in a cancerous state. In the present specification, examples of cancer cells include melanoma, lung cancer, stomach cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, thymic cancer, lymphoma, sarcoma, liver cancer, non-Hodgkin Lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer (colorectal cancer), multiple myeloma, neuroblastoma, bladder cancer And cells such as cervical cancer, skin cancer, breast cancer, esophageal cancer, nephroma, and brain tumor, but are not limited thereto.

  As used herein, “cancer treatment” includes surgical treatment performed by administration of an anticancer agent (eg, chemotherapeutic agent, radiotherapy, etc.) or surgical removal. It is understood that any cancer treatment can be used when diagnosed with cancer according to the present invention.

  The chemotherapeutic agents used in the present specification are well known in the art, and are described in the anticancer agent manual second edition Shigeru Tsukakoshi et al., Chugai Medical Co .; Pharmalogy; Lippincott Williams & Wilkins, Inc. It is described in. Examples of such chemotherapeutic agents include, but are not limited to, the following: 1) alkylating agents (alkylate cellular components such as DNA and proteins to show cytotoxicity. For example, cyclophosphamide, busulfan 2) Antimetabolites (mainly agents that inhibit nucleic acid synthesis (eg, methotrexate as an antifolate), 6-mercaptopurine as a purine antimetabolite Such as fluorouracil (5-FU) as a pyrimidine antimetabolite); 3) DNA topoisomerase inhibitors (eg, camptothecin, etoposide (inhibits topoisomerase I and II, respectively)); 4) tubulin action Drug (inhibits microtubule formation and suppresses cell division. Vinblastine, 5) platinum compound (shows cytotoxicity due to binding to DNA and protein; cisplatin, carboplatin, etc.); 6) anticancer antibiotic (binds to DNA and inhibits DNA synthesis and RNA synthesis; adriamycin. Daunorubicin, mitomycin C, bleomycin, etc.); 7) Hormonal agents (adapted to hormone-dependent cancers such as breast cancer, uterine cancer, prostate cancer, tamoxifen, leuprorelin (LH-RH), etc.); 8) organisms Formulations (including asparagine effective against asparagine-requiring hematological malignancy, interferon showing direct antitumor action and indirect action by immune enhancement); 9) Immunostimulant (enhance immune response ability) Indirect antitumor activity: Lentinan, a polysaccharide derived from shiitake mushroom, Such as bestatin is a tide).

  As used herein, the term “anticancer agent” selectively suppresses the growth of cancer (tumor) cells, and includes both cancer drugs and radiation therapy. Such anticancer agents are well known in the art, and are described, for example, in the anticancer agent manual 2nd edition Shigeru Tsukagoshi et al., Chugai Medical Co .; Pharmalogy; Lippincott Williams & Wilkins, Inc. It is described in.

  As used herein, “radiotherapy” or “radiotherapy” are used interchangeably and refer to treatment of a disease utilizing ionizing radiation or a radioactive substance. Typical radiotherapy includes, but is not limited to, X-rays, γ-rays, electron beams, proton beams, heavy particle beams, and neutron capture therapy. Preferable radiation therapy includes heavy particle beam. The therapy using heavy particle beams may be uncommon due to the large apparatus. Such radiation therapy is well known in the art, for example, the basics of radiation examination and treatment; radiation therapy and multidisciplinary treatment: Kei Keizen (Shiga Medical University, Radiation): General Gastroenterology Care, Vol. 6, No. 6, Page 79-89, 6-7 (2002.02).

(Specific factor)
As used herein, “specifically interacts” with a polynucleotide or polypeptide means that the affinity for the polynucleotide or polypeptide is other unrelated (especially less than 30% identity). ) Refers to typically equivalent or higher, preferably significantly higher, than the affinity for the polynucleotide or polypeptide. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like. Typically, the specific interaction can be determined by whether it can be detected by Western blotting. Thus, “not interacting” as used herein includes, but is not limited to, not being detected significantly in Western blots.

  As used herein, “factor” may be any substance or other element (for example, energy) as long as the intended purpose can be achieved. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), wherein the polypeptide is a receptor. -Or specific ligand or receptor when it is a ligand; and when the polypeptide is an enzyme, include but are not limited to its substrate.

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, a low molecular weight organic material has a molecular weight of about 1000 or less, but may have a higher molecular weight. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). It is not limited.

(Diagnosis, treatment, prevention)
As used herein, “diagnosis” refers to identifying various parameters related to the type of disease, type and degree of disorder, physical condition, etc. in the subject, Determining responsiveness prediction, pathological change prediction or cause. By using the methods, devices, and systems of the present invention, a disease can be analyzed and correlated with a disease state, and such information can be used to identify the disease, disorder, condition, drug to be administered in a subject. Various parameters such as formulation or method for treatment or prevention such as type and amount can be selected.

  The diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method can be used from the body and can be carried out away from the hands of medical personnel such as doctors.

  As used herein, “high risk” means that when used for a disease, the subject is likely to have the disease (eg, about 25%, about 50%, about 75%, etc.). ). A subject who has received a high-risk diagnosis can make a definitive diagnosis by another diagnostic method.

  As used herein, “determined diagnosis”, when used for a certain disease, refers to a definite diagnosis that a subject suffers from the disease. The present invention is mainly used as early diagnosis and comprehensive diagnosis. Therefore, in such a definitive diagnosis, a definitive diagnosis of cancer can be performed by using a marker specific to a specific cancer and / or histological examination.

  As used herein, “treatment” refers to preventing a disease or disorder from deteriorating, preferably maintaining the status quo, more preferably reducing, when a certain disease or disorder becomes such a condition. Preferably, it means that it is reduced.

  As used herein, “prevention” includes preventing a certain disease or disorder from being brought into such a state, delaying, and worsening.

  In the present specification, the “instruction” is a description of a method of using the present invention or a method of diagnosis for a doctor, a person who performs administration such as a subject, and a person who diagnoses (which may be the subject). is there. When a diagnostic agent or the like is provided as a kit, instructions can be attached to instruct how to use the instruction. This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided in a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page provided on the Internet (web) Site), e-mail, SMS, PDF document, etc.).

(screening)
As used herein, “screening” refers to selecting a target such as an organism, cell, or substance having a specific target property from a large number of populations by a specific operation / evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. For the screening, a system using an actual substance such as in vitro or in vivo may be used, or a library generated using an in silico system (a system using a computer) may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing drugs, diagnostic agents, therapeutic agents, etc. by computer modeling based on the disclosure of the present invention.

(Gene therapy)
In certain embodiments, a nucleic acid comprising a normal or variant of a nucleic acid sequence that encodes a protein of the invention, a sequence that encodes an antibody or functional derivative thereof, is capable of treating a disease or disorder associated with a malignant tumor. Administered for gene therapy purposes to treat, inhibit or prevent. Gene therapy refers to a therapy performed by administering to a subject a nucleic acid that is expressed or expressible. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

  Any method for gene therapy available in the art can be used in accordance with the present invention. Exemplary methods are a general overview of gene therapy methods, Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev , Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual. , Stockton Press, NY (1990).

(Description of Preferred Embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(New protein of the present invention)
The protein of the present invention is a protein that interacts specifically with an anti-PCNA polyclonal antibody and has a molecular weight larger than PCNA on SDS-PAGE (also referred to herein as “PCNA-related antigen”) or a modification thereof About the body. Here, the molecular weight recognized by a polyclonal antibody against PCNA (proliferating cell nuclear antigen) (Santa Cruz, USA) is 36000 to 43500 daltons, preferably 3600 to 40000 daltons, more preferably 36000 to 38000 daltons, and more preferably about 37000 daltons It is a protein. PCNA is a non-histone acidic nucleoprotein with a molecular weight of 35,000 to 36000, and apparently is not the same protein because of the different molecular weights on SDS-PAGE.

  As used herein, “PCNA” is an abbreviation for PROLIFERATING CELL NUCLAR ANTIGEN (PCNA), and is also referred to as DNA POLYMERASE DELTA AUXILARY PROTEIN (Biochemistry Vol. 68, 1542-1548). (Known in 1996) as a replication protein that functions as a cofactor for pol δ). Travali et al. Isolated the full-length human PCNA gene and its flanking cDNA in 1989 (Travali, S. et al., J. Biol. Chem. 264: 7466-7472, 1989). PCNA is associated with ubiquitin, a small ubiquitin-related modifier (SUMO), and ubiquitin and SUMO compete with PCNA modifications. Thus, PCNA is considered an essential factor for the progress of DNA replication and repair.

  The sequence of PCNA is typically described in SEQ ID NO: 1 (nucleic acid) and SEQ ID NO: 2 (amino acid). However, there is a strong possibility that it is at least a PCNA-related antigen or a variant thereof in that it reacts with a polyclonal antibody. PCNA antigens were first discovered as nuclear antigens that react with antibodies in the serum of subjects with systemic lupus erythematosus (SLE), an autoimmune disease (Miyachi K. et al., J. Immun. 121,2228-2234,1978). Subsequently, it has been reported that DNA damage is removed and repaired (ShivjiM. Et al., Cell 69,367-374, 1992) and the cell cycle regulatory mechanism is involved (Xiong Cell, 505-514, 1992). PCNA has been shown to provide a molecular surface with various functions by forming a trimer, but its interaction with other molecules is unknown and useful as a tumor marker. The nature is also not clear (Hiroshi Morioka, Biochemistry Vol. 68, No. 9, 1542-1548, 1996).

For PCNA, reference is also made to the following.
1. Bravo, R., Exp. Cell Res. 163: 287-293, 1986.
2. Hasan, S. et al., Nature 410: 387-391, 2001.
3. Hoege, C. et al., Nature 419: 135-141, 2002.
4. Ku, D.-H. et al., Genet. 15: 297-307, 1989.
5. Mann, MJ et al., Proc. Nat. Acad. Sci. 92: 4502-4506, 1995.
6. Rao, VVNG et al., Cell Genet. 56: 169-170, 1991.
7. Stelter, P. et al., Nature 425: 188-191, 2003.
8. Suzuka, I. et al., Proc. Nat. Acad. Sci. 86: 3189-3193, 1989.
9. Taniguchi, Y. et al., Mammalian Genome 7: 906-908, 1996.
10. Travali, S. et al., J. Biol. Chem. 264: 7466-7472, 1989.
11. Webb, G. et al., Genet. 86: 84-86, 1990.
The PCNA-related antigen is proliferated remarkably in the serum of malignant tumor subjects such as cancer, and its appearance frequency is specific to cancer, etc. Is useful. Such usefulness is an effect provided for the first time in the present invention.

(Method for producing the protein of the present invention)
Examples of the method for producing the protein of the present invention include chemical separation and purification methods and gene recombination methods.

  As the chemical separation and purification method, for example, commercially available high performance liquid chromatography and ion exchange chromatography can be used. The method is described in detail in “Nippon Analytical Chemistry, Kanto Branch, High Performance Liquid Chromatography Handbook, Maruzen, 1985”.

  As a genetic recombination method, for example, a recombinant vector is constructed by inserting a DNA encoding the protein of the present invention into a vector, and this is inserted into a host to produce a transformant. There is a method for purifying a target protein from

  Transformants derived from microorganisms, animal cells, etc. that possess recombinant vectors incorporating the DNA encoding the polypeptide of the present invention are cultured according to a normal culture method to produce the polypeptide of the present invention. The polypeptide of the present invention can be produced by accumulating and collecting the polypeptide of the present invention from the culture of the present invention.

  The method of culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism of the present invention. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

  Examples of vectors include plasmids and fuzzy.

  Examples of the host include Escherichia coli, Bacillus subtilis, and yeast.

  Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing them, starch or starch hydrolysate carbohydrates, acetic acid, propionic acid Organic acids such as ethanol, alcohols such as ethanol and propanol can be used.

  Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing substances, peptone, meat extract, yeast extract, and corn-prica. −, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof, and the like can be used.

  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

  The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin or tetracycline, to a culture medium as needed during culture | cultivation.

  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is transformed with an expression vector using trp promoter. When cultivating the microorganism, indoleacrylic acid or the like may be added to the medium. Plant cells or organs into which a gene has been introduced can be cultured in large quantities using a jar fermenter. As the culture medium, a commonly used Murashige and Skug (MS) medium, White medium, or a medium in which plant hormones such as auxin and cytokinin are added to these mediums, etc. are used. Can do.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention may be RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122 , 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Proceedings of the Society for the Biological Medicine, 73, 1 (1950)] or fetal bovine serum or the like was added to these mediums. A medium or the like is used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  In order to isolate or purify the polypeptide of the present invention from the culture of the transformant transformed with the nucleic acid sequence encoding the polypeptide of the present invention, a conventional enzyme well-known in the art may be used. Isolation or purification methods can be used. For example, when the polypeptide of the present invention is secreted outside the transformant for producing the polypeptide of the present invention, the culture is treated with a technique such as centrifugation to obtain a soluble fraction. Get minutes. From the soluble fraction, anion exchange chromatography using a resin such as a solvent extraction method, a salting out method using ammonium sulfate, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), etc. Chromatography method, cation exchange chromatography method using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using a resin such as butyl sepharose or phenyl sepharose, gel filtration using a molecular sieve A purified sample can be obtained using a method such as electrophoresis, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing.

  When the polypeptide of the present invention accumulates in a dissolved state in the cells of the transformant for producing the polypeptide of the present invention, the culture is centrifuged to collect the cells in the culture and wash the cells. After that, the cells are crushed with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical Co., Ltd.) Anion exchange chromatography using resin, cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose, phenyl sepharose The purified sample is obtained by using a method such as a hydrophobic chromatography method, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, and an electrophoresis method such as isoelectric focusing. be able to.

  When the polypeptide of the present invention is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted and centrifuged from the precipitate fraction obtained by the usual method. After recovering the polypeptide, the insoluble body of the polypeptide is solubilized with a polypeptide denaturant. The solubilized solution is diluted or dialyzed into a dilute solution that does not contain the polypeptide denaturing agent or the concentration of the polypeptide denaturing agent does not denature the polypeptide. Then, a purified sample can be obtained by the same isolation and purification method as described above.

  Further, the protein can be purified according to a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458]. In addition, the polypeptide of the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having an affinity for the fused protein [Akio Yamakawa, Experimental Medicine (Experimental Medicine), 13, 469-474 (1995)]. For example, according to the method described by Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227-8231 (1989), Genes Develop., 4, 1288 (1990)] The peptide can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

  In addition, the polypeptide of the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989). ), Genes Develop., 4,1288 (1990)].

  Furthermore, it can also be purified by affinity chromatography using an antibody against the polypeptide itself of the present invention. The polypeptide of the present invention is produced in vitro according to a known method [J. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)]. Can be produced using transcription and translation systems.

  Based on the amino acid information of the polypeptide obtained above, the polypeptide of the present invention can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). can do. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

  The structural analysis of the purified polypeptide of the present invention is carried out by a method usually used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, published by Tokyo Kagaku Dojin, 1993). Is possible. The physiological activity of the novel ps20-like polypeptide of the present invention can be determined by a known assay [Cell, 75, 1389 (1993), J. Cell Bio. L146, 233 (1999), Cancer Res. 58,1238 (1998), Neuron. 17, 1157 (1996), Science 289, 1197 (2000)].

  Deletion, substitution or addition of amino acids of the polypeptide of the present invention can be carried out by site-directed mutagenesis which is a well-known technique. Such deletion, substitution or addition of one or several amino acids is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons ( 1987-1997), Nucleic Acids Research, 10,6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 ( 1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), Proc. Natl. Acad. Sci., USA, 81, 5662 (1984), Science, 224, 1431 (1984), PCT WO85 / It can be prepared according to the method described in 00817 (1985), Nature, 316, 601 (1985).

(Factors that specifically interact with the protein of the present invention)
In another aspect, the present invention provides an agent that specifically interacts with an anti-PCNA polyclonal antibody that specifically interacts with a protein having a higher molecular weight on SDS-PAGE than PCNA. This factor can be, but is not limited to, nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof. Preferably, it may be an antibody or a derivative thereof. Antibodies are preferred. In particular, a monoclonal antibody is more preferable, and it is further preferable that the monoclonal antibody does not cross-react with PCNA. This is because, by using an antibody that does not cross-react with PCNA, cancer can be easily diagnosed with a dot blot or the like, but is not limited thereto.

  Therefore, the factor of the present invention can be used as a probe. Such probes are useful in assays using immune responses.

  In one embodiment, preferably the agent of the invention is labeled or can be labeled. This is because it can be used in an assay. It is understood that being able to be labeled refers to a substance whose presence can be detected, for example, by adding it to a factor and using another second factor. It is understood that the labeling can use mechanisms such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen-antibody reaction.

  In another aspect, the present invention provides a hybridoma that produces the factor (preferably an antibody) of the present invention. It will be appreciated that hybridoma production methods may employ techniques well known in the art, as detailed herein below.

  The production of antibodies that recognize the polypeptides of the present invention is also well known in the art. For example, a polyclonal antibody is prepared by administering a full-length or partial fragment purified preparation of the obtained polypeptide or a peptide having a partial amino acid sequence of the protein of the present invention as an antigen to an animal. Can do.

  When producing an antibody that is a representative example of the factor of the present invention, rabbits, goats, rats, mice, hamsters and the like can be used as animals to be administered. The dose of the antigen is preferably 50 to 100 μg per animal. When a peptide is used, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin as an antigen. The peptide used as an antigen can be synthesized with a peptide synthesizer. The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. On the 3rd to 7th day after each administration, blood was collected from the fundus venous plexus, and the serum reacted with the antigen used for immunization. [Enzyme immunoassay (ELISA method): 1976, published by Medical Shoin, Antibodies-A Laboratory Manual, Cold Spring Harbor Lavoratory (1988)].

  Serum can be obtained from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and polyclonal antibodies can be separated and purified from the serum using well-known techniques. The production of monoclonal antibodies is also well known in the art. For the preparation of antibody-producing cells, first, a rat whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the polypeptide of the present invention used for immunization was used as a source of antibody-producing cells. A hybridoma is prepared by fusion with myeloma cells. Thereafter, a hybridoma that specifically reacts with the partial fragment polypeptide of the polypeptide of the present invention is selected by enzyme immunoassay or the like. The monoclonal antibody produced from the hybridoma thus obtained can be used for various purposes.

  The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, by chemical synthesis, or preferably by recombinant expression techniques.

  Recombinant expression of an antibody of the invention, or a fragment, derivative or analog thereof (eg, a heavy or light chain of an antibody of the invention or a single chain antibody of the invention) contains a polynucleotide that encodes the antibody. It is necessary to construct an expression vector. Once a polynucleotide encoding the antibody molecule of the invention or the heavy or light chain of an antibody, or part thereof (preferably containing the variable domain of the heavy or light chain) is obtained, the antibody molecule Vectors for the production of can be generated by recombinant DNA techniques using techniques well known in the art. Thus, methods for preparing proteins by expression of polynucleotides containing nucleotide sequences encoding antibodies are described herein. Methods well known to those skilled in the art can be used for the construction of expression vectors containing sequences encoding antibodies and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention includes an antibody molecule of the present invention, or a heavy or light chain thereof, or a nucleotide sequence that encodes a heavy or light chain variable domain, operably linked to a promoter. Provide a replicable vector. Such vectors may comprise a nucleotide sequence that encodes the constant region of the antibody molecule (see, eg, PCT Publication WO86 / 05807; PCT Publication WO89 / 01036; and US Pat. No. 5,122,464). , And the variable domains of the antibody can be cloned into such vectors for the entire expression of heavy or light chains.

  This expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibodies of the invention. Accordingly, the present invention provides a host cell comprising an antibody of the invention, or a heavy or light chain thereof, or a polynucleotide encoding a single chain antibody of the invention, operably linked to a heterologous promoter. including. In a preferred embodiment for the expression of double chain antibodies, vectors encoding both heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule.

  Such an antibody can be used, for example, in the immunological detection method of the polypeptide of the present invention. As the immunological detection method of the polypeptide of the present invention using the antibody of the present invention, as necessary, Examples thereof include an ELISA method using a microtiter plate, a fluorescent antibody method, a Western blot method, an immunohistochemical staining method, and the like.

It can also be used in an immunological quantification method of the polypeptide of the present invention. As a method for quantifying the polypeptide of the present invention, a sandwich ELISA method using two kinds of monoclonal antibodies having different epitopes among antibodies that react with the polypeptide of the present invention in a liquid phase, and a radioisotope such as ¹²⁶ I are used. And a radioimmunoassay method using a protein of the present invention labeled with a body and an antibody recognizing the protein of the present invention.

  Methods for quantifying mRNA of the polypeptide of the present invention are also well known in the art. For example, using the above-mentioned oligonucleotide prepared from the polynucleotide or DNA of the present invention, the expression level of DNA encoding the polypeptide of the present invention is expressed at the mRNA level by the Northern hybridization method or the PCR method. It can be quantified. Such techniques are well known in the art and are also described in the literature listed herein.

  These polynucleotides can be obtained and the nucleotide sequence of these polynucleotides can be determined by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, Kutmeier et al., BioTechniques 17: 242 (1994) In brief) synthesis of overlapping nucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then Amplification of this linked oligonucleotide by PCR.

  The polynucleotide encoding the antibody can be made from nucleic acid from a suitable source. Although a clone containing nucleic acid encoding an antibody is not available, if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized, or appropriate From any source (eg, an antibody cDNA library, or any tissue or cell expressing an antibody (eg, a hybridoma cell selected for expression of an antibody of the invention), or from there In order to identify a cDNA clone from an isolated nucleic acid (preferably poly A + RNA), for example from a cDNA library encoding the antibody, it hybridizes to the 3 'and 5' ends of the sequence. Use an oligonucleotide probe specific for that particular gene sequence by PCR amplification using possible synthetic primers B - it may be obtained by training. Amplified nucleic acids generated by PCR can be cloned into replicable cloning vectors using any method known in the art.

  Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined, the nucleotide sequence of the antibody can be determined using methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, eg, Sambrook et al., Supra and Ausubel et al., Supra, techniques described above, both of which are incorporated herein by reference in their entirety.) For example, antibodies having different amino acid sequences can be made to generate amino acid substitutions, deletions, and / or insertions.

  In certain embodiments, the heavy chain variable domain and / or light chain variable domain amino acid sequence is determined by methods well known in the art for identification of complementarity determining region (CDR) sequences (eg, sequence hypervariables). To determine the sex region, it can be examined (by comparison with known amino acid sequences of other heavy and light chain variable regions). Using conventional recombinant DNA technology, one or more CDRs can be placed in the framework region as described above (eg, in the human framework region to humanize non-human antibodies). Can be inserted). This framework region can be naturally occurring or can be a consensus framework region and preferably can be a human framework region (eg, the listed human framework regions). (See Chothia et al., J. Mol. Biol. 278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of the framework region and the CDR codes for an antibody that specifically binds to the polypeptide of the present invention. Preferably, as discussed above, one or more amino acid substitutions can be made within the framework region, and preferably the amino acid substitution improves the binding of the antibody to its antigen. . In addition, such methods also provide for amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Can be used to make. Other changes to the polynucleotide are encompassed by the present invention and in the art.

  In addition, a technique developed for the production of “chimeric antibodies” (Morrison) by splicing genes from appropriate antigen-specific mouse antibody molecules with genes from human antibody molecules of appropriate biological activity. et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985) ) Can be used. As described above, a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from mouse mAb and human immunoglobulin constant regions (eg, humanized antibodies). .

  When producing single chain antibodies, known techniques described for the production of single chain antibodies (US Pat. No. 4,946,778; Bird, Science 242: 423-42 (1988); Huston etal., Proc. Natl Acad. Sci. USA 85: 5879-5883 (1988); and Ward et al., Nature 334: 544-54 (1989)) can be utilized. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

(Diagnostic composition)
In another aspect, the present invention provides a composition for diagnosis of a malignant tumor, comprising a protein that specifically interacts with an anti-PCNA polyclonal antibody and has a higher molecular weight on SDS-PAGE than PCNA. To do. A composition containing such a protein has not been conventionally known, and since this protein is universally expressed in malignant tumor subjects and not in normal subjects, it is used as a diagnostic marker. It is understood that is useful. Such a composition can be used as a standard substance of a diagnostic reagent when diagnosing a malignant tumor. Typically, a radioimmunoassay (RIA) or ELISA method can be used. In this case, it can be determined by preparing an antibody as a factor specific to this protein and measuring the antigen using the antibody. As the measuring method, any method as described in this specification can be used. It will be appreciated that the proteins of the present invention can take any form as described herein in the section (Novel proteins of the present invention).

  In another aspect, the present invention includes a malignant agent comprising a factor that specifically interacts with a protein having a molecular weight on SDS-PAGE larger than PCNA, which specifically interacts with an anti-PCNA polyclonal antibody. Compositions for tumor diagnosis are provided. The composition containing the factor of the present invention can be used for diagnosis of a malignant tumor by detecting the protein of the present invention. For such detection, any immunological technique can be used. For example, ELISA, RIA, EIA, RIBA and the like can be used, but are not limited thereto. Therefore, the factor of the present invention is advantageously highly specific. In particular, it is preferable to react with PCNA in a distinguishable manner. This is because there is no need to sort by molecular weight.

  It will be understood that the factors of the present invention can take any form as described herein (factors that specifically interact with the novel proteins of the present invention).

(Measurement methods such as cancer)
In another aspect, the present invention provides a method for diagnosing a malignant tumor in a subject, comprising: (A) obtaining a sample from the subject; (B) the sample and an anti-PCNA polyclonal antibody A step of mixing a factor that specifically interacts with a protein that specifically interacts with a protein having a molecular weight on SDS-PAGE larger than PCNA; (C) the amount of interaction between the sample and the factor And (D) determining whether the interacted amount is greater than the amount of a healthy person. Here, malignant tumors include bile duct cancer, melanoma, lung cancer, stomach cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymic cancer, lymphoma, sarcoma, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, breast cancer (chest cancer), prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer (colorectal cancer), multiple myeloma, neuroblastoma, bladder Can diagnose any malignant tumor such as cancer, cervical cancer, skin cancer, breast cancer, esophageal cancer, nephroma, brain tumor, osteosarcoma, Ewing sarcoma, and malignant fibrohistiocytoma Is understood.

  In a preferred embodiment, the malignant tumor is from bile duct cancer, prostate cancer, stomach cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, osteosarcoma, Ewing sarcoma and malignant fibrohistiocytoma. At least one tumor selected from the group consisting of: More preferably, the malignant tumor consists of bile duct cancer, prostate cancer, stomach cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, osteosarcoma, Ewing sarcoma, and malignant fibrohistiocytoma Includes all tumors selected from the group. There has been no marker that can comprehensively diagnose and detect such a wide variety and a wide variety of malignant tumors, and the present invention is revolutionary in that it provides a comprehensive cancer marker and a marker for malignant tumors. It can be said that there is a great effect.

  Such a cancer detection method can be typically performed by Western blotting (Harlow et al Cold Spring Harbor Lab, 471-510, 1988). For example, a method in which a sample is electrophoresed and separated (electrophoresis), an antibody labeled with a label specific to the protein to be detected is used, and the protein in the sample is measured using the label ( Immunoassay) can be used. As the immunoassay, an enzyme immunoassay in which the label is an enzyme, a radioimmunoassay in which the label is a radioisotope, or the like can be used. In the present invention, “detection” includes not only qualitative measurement but also quantitative measurement such as “measurement”.

  In another embodiment, in the present invention, as a method for measuring the present protein using an enzyme immunoassay, for example, a monoclonal antibody against the present protein is used as an antibody antibody-producing cell and a myeloma cell. It can be prepared by a cell fusion method and performed by an immunoantigen antibody method using this monoclonal antibody. For example, one of the monoclonal antibodies is immobilized on a carrier, a sample is added to the carrier, an enzyme-labeled monoclonal antibody is added, washed, a substrate is added, and the enzyme and the substrate are reacted. A method of measuring luminescence, color development, etc. by the reaction can be used.

  As the carrier, for example, a microtiter plate or latex particles can be used.

  Examples of the method for fixing the monoclonal antibody to the carrier include a binding method using biotin and avidin (or streptavidin). A general method of an immobilization method using biotin and avidin (or streptavidin) is described in, for example, “Eiji Ishikawa, Ultrasensitive Enzyme Immunoassay, Academic Publishing Center, 1993”.

  As the sample used in the detection / diagnosis of the present invention, for example, serum, urine, sweat, lymph, plasma, blood, saliva, etc. of the subject can be used. As a cleaning solution used for cleaning, for example, a phosphate buffer containing a surfactant can be used. As the phosphate buffer, for example, Dulbecco's PBS, such as Mg, can be used.

  As the substrate, for example, when the enzyme is peroxidase, 3,3 ', 5,5'-tetramethylbenzidine (manufactured by Sigma, trade name: TMB) can be used.

  As a method for measuring cancer or the like of the present invention, for example, a method is used in which the serum of a healthy person and the serum of a subject are used as serum, the target protein in the subject serum is measured, and the results are compared. can do.

  The serum of healthy people does not contain this protein. Therefore, when the serum of a healthy person is used, the protein of the present invention is not detected. However, this protein is expressed at a high rate in the serum of malignant tumor subjects such as cancer. Therefore, it can be diagnosed that there is a possibility that the subject is a malignant tumor such as cancer, based on the expression of this protein observed using the serum of the subject. In addition, when actually diagnosing which organ is a malignant tumor, it can be used in combination with other known organ-specific cancer diagnostic methods and taking into account the results. preferable.

(Diagnostic agent / kit)
In another aspect, the present invention provides a kit for diagnosing a malignant tumor in a subject, wherein (A) the molecular weight on SDS-PAGE specifically interacts with an anti-PCNA polyclonal antibody is PCNA. Providing a kit, wherein the appearance or increase of expression of the protein indicates that the subject has or is likely to have a malignant tumor To do. Here, the factor of the present invention is usually used as a means for detecting the protein of the present invention. The protein of the present invention can be detected by using such a factor and using a label or the like as necessary. Based on the amount of protein detected by the assay, it is understood how much the protein of the present invention is expressed and can be used for diagnosis of the presence or absence of malignant tumor by comparing with the normal value. Such a kit is sometimes referred to herein as a diagnostic agent.

  The diagnostic agent of the present invention comprises a protein (PCNA-related antigen) having a molecular weight of more than 36000 (for example, 36000 to 43500 daltons, preferably 36000 to 40000 daltons, more preferably 36000 to 38000 daltons, more preferably 37000 daltons), or Mutant antigen) as an active ingredient, for example, a carrier on which a monoclonal antibody against the protein of the present invention is immobilized. In addition to the carrier, enzyme-labeled monoclonal antibody, substrate, antiseptic And a kit in which an agent, a stabilizer, a sensitizer and the like are enclosed.

  This protein is markedly increased in the serum of subjects with malignant tumors of non-epithelial origin such as osteosarcoma and malignant fibrous histiocytoma as well as solid tumors. However, in view of the fact that it is not expressed at all in the serum of a healthy person as a control group, a test using the expression of this protein as a marker is extremely significant as a clinical biochemical and clinicopathological diagnostic method.

  Most of the conventional cancer diagnostic markers correspond to only one organ or organ, and thus the significance of developing diagnostic techniques that can monitor the presence or absence of malignant tumors throughout the body in a single examination is extremely significant. As a primary screening, it can be applied to daily health examinations.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

  The reagents used in the examples shown below were obtained from Wako Pure Chemicals, Sigma unless otherwise stated. Animal breeding, National Society for Medical Research have created "Principles of Laboratory Animal Care" and the Institute of Laboratory Animal Resource is created, National Institute of Health has published "Guide for the Care and Use of Laboratory Animals" (NIH Publication , No. 86-23, 1985, revised) in accordance with the spirit of animal welfare. When human subjects were used, experiments were conducted after obtaining prior consent.

(Example 1: Measurement of cancer, etc.)
In this example, patient sera of malignant tumors such as cancer that were collected by volunteers and obtained normal human serum and informed consent were employed as subjects. Serum was used as it was at 30-fold dilution. Was used to assay malignant tumors such as cancer.

  The subject serum was electrophoresed on SDS-PAGE (polyacrylamide gel electrophoresis) according to the manufacturer's manual, Biorad, for electrophoresis and transfer, and a nylon membrane (Millipore: Bedford, MA, 01730, USA). The membrane was blocked overnight at 4 ° C. with Blockace (trade name Dainippon Pharmaceutical, Osaka), and a commercially available rabbit anti-PCNA polyclonal antibody (FL-261: sc-7907; Santa Cruz Biotechnology, Inc.) was used. It reacted at room temperature for 1 hour. The plate was washed 5 times for 5 minutes each with PBS / 0.1% Tween-20 (surfactant) (Nacalai tesque, Kyoto, Japan). After this, HRPO (hoe radish peroxydase) -labeled anti-rabbit IgG (serum) (Goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc 2145 Delaware Avenue, Santa Cruz, 50 hours) was added. Then, it was washed with 10 mM Tris-HCL (pH 7.4) -0.14 M NaCl-0.1% Tween 20 (buffer) 5 times for 5 minutes, respectively, followed by 10 mM Tris-HCL (pH 7.4). Each was washed twice for 5 minutes, developed with a commercially available ECL reagent, exposed to X-ray film, and the molecular weight on SDS-PAGE was larger than 36000 Dalton and smaller than 43500. In particular, it was positive with the expression of a reaction line of about 37,000 daltons (FIG. 1) As an antibody, PCNA (C-20): sc-9857 sold by the same Santa Cruz Biotechnology: MS- sold by Funakoshi The same detection can be performed using catalog numbers such as 106-P0 and MS-106-R7.

  The results are shown in the table below.

(table)
Solid cancer Bile duct cancer 1/2 Expression in 1 sample Prostate cancer 2/2 2 expression in 2 samples Gastric cancer 3/4 3 expression in 4 samples Breast cancer 7/10 7 Expression in 10 samples Esophageal cancer 12/18 12 specimens in 18 specimens liver cancer 2/4 2 specimens in 4 specimens colorectal cancer 4/4 4 specimens in 4 specimens subtotal 31/44 31 specimens in 44 specimens sarcoma osteosarcoma 2/3 3 2 expression cases in specimen malignant fibrohistiocytoma 3/3 3 expression cases in 3 specimens Subtotal 5/7 5 expression cases in 7 specimens

Total 36/51 36 cases in 51 specimens

Healthy subjects 0/30 No expression in 30 samples

As is apparent from the table, this protein is markedly increased not only in solid cancer but also in the serum of non-epithelial malignant tumors such as osteosarcoma and malignant fibrous histiocytoma. However, in view of the fact that it is not expressed at all in the serum of a healthy person as a control group, a test using the expression of this protein as a marker is extremely significant as a clinical biochemical and clinicopathological diagnostic method. Most of the conventional cancer diagnostic markers correspond to only one organ / organ, and thus the development of diagnostic technology that can monitor the presence or absence of malignant tumors throughout the body in a single examination is extremely significant. As a primary screening, it can be applied to daily health examinations.

(Example 2: Purification of the protein of the present invention)
Rabbit thymus acetone powder was stirred overnight at 4 ° C. After centrifugation, the supernatant was applied by Western blotting. As a result, the protein had the same molecular weight as that of a protein having a molecular weight of 3600 to 43500 dalton (approximately 37,000 dalton) expressed in the serum of a malignant tumor specimen such as cancer, and anti-PCNA We were able to find a positive protein with the antibody. By further purifying this by high performance liquid chromatography and ion exchange chromatography, it is considered that a highly pure protein can be obtained. This protein having a molecular weight of 36,000 to 43500 dalton (approximately 37,000 daltons) is a standard substance for this diagnostic reagent, and has a great profit from mass production. Although this protein is a substance produced in malignant tumors, it is a substance that can always be obtained from rabbit thymus using this molecular weight as an index, and it is not necessary to select accidental, limited and non-purpose means from malignant tumor cells. . Therefore, the present invention allows considerable clinical application by a planned and effective means.

(Example 3: Production of antibodies specific to the protein of the present invention)
Next, an antibody that does not interact with PCNA but interacts with the protein of the present invention is prepared. Such an antibody is produced by purifying this protein, using it to immunize an animal such as a rabbit or rat, and then selecting an antibody obtained from a strain that does not interact with PCNA. can do. Therefore, by using such an antibody, cancer or the like can be diagnosed without performing separation by molecular weight by a method such as dot blot.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, malignant tumors can be comprehensively diagnosed by a simple method. Accordingly, the industrial use of the present invention is found, for example, in the pharmaceutical industry.

FIG. 1 shows the results of Western blot experiments using anti-PCNA antibodies after electrophoresis using samples from various cancer patients.

(Explanation of sequence listing)
SEQ ID NO: 1 is the nucleic acid sequence of PCNA.
SEQ ID NO: 2 is the amino acid sequence of PCNA.

Claims (22)

A protein having a larger molecular weight on SDS-PAGE or a variant thereof, which specifically interacts with an anti-PCNA polyclonal antibody. 2. The protein of claim 1, wherein the molecular weight is greater than 36000 daltons and less than 43500 daltons. The protein of claim 1, wherein the molecular weight is greater than 36000 daltons and less than 40000 daltons. The protein of claim 1, wherein the molecular weight is greater than 36000 daltons and less than 38000 daltons. The protein according to claim 1, wherein the PCNA has an amino acid sequence represented by SEQ ID NO: 2. An agent that specifically interacts with an anti-PCNA polyclonal antibody and that specifically interacts with a protein having a molecular weight larger than PCNA on SDS-PAGE. The factor according to claim 6, wherein the factor is selected from the group consisting of a nucleic acid molecule, a polypeptide, a lipid, a sugar chain, a small organic molecule, and a complex molecule thereof. The factor according to claim 6, wherein the factor is an antibody or a derivative thereof. The factor according to claim 6, wherein the factor is used as a probe. 7. The agent of claim 6, wherein the agent is labeled or can be labeled. 7. The agent of claim 6, wherein the label utilizes a technique selected from the group consisting of fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction and antigen-antibody reaction. 7. A factor according to claim 6, which does not interact with PCNA. 7. A factor according to claim 6, which is a monoclonal antibody. The factor according to claim 6, wherein the PCNA has the amino acid sequence shown in SEQ ID NO: 2. A hybridoma which produces the factor according to claim 13. A composition for diagnosis of a malignant tumor, comprising a protein that specifically interacts with an anti-PCNA polyclonal antibody and has a molecular weight on SDS-PAGE larger than that of PCNA. A composition for diagnosis of a malignant tumor, comprising a factor that specifically interacts with an anti-PCNA polyclonal antibody and specifically interacts with a protein having a molecular weight on SDS-PAGE larger than that of PCNA. A kit for diagnosing a malignant tumor in a subject,
(A) Means for detecting a protein that interacts specifically with an anti-PCNA polyclonal antibody and has a molecular weight larger than PCNA on SDS-PAGE;
With
Here, the appearance or increase of the expression of the protein indicates that the subject has or is suspected of having a malignant tumor,
kit. A method for diagnosing a malignant tumor in a subject comprising:
(A) obtaining a sample from the subject;
(B) a step of mixing the sample with a factor that specifically interacts with an anti-PCNA polyclonal antibody and specifically interacts with a protein having a molecular weight on SDS-PAGE larger than that of PCNA;
(C) measuring the amount of interaction between the sample and the factor;
(D) determining whether the amount of interaction is greater than the amount of healthy individuals;
Including the method. The malignant tumors include bile duct cancer, melanoma, lung cancer, gastric cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, thymic cancer, lymphoma, sarcoma, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma , Leukemia, uterine cancer, breast cancer (chest cancer), prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer (colorectal cancer), multiple myeloma, neuroblastoma, bladder cancer, 20. The method of claim 19, wherein the method is selected from cervical cancer, skin cancer, breast cancer, esophageal cancer, nephroma, brain tumor, osteosarcoma, Ewing sarcoma, and malignant fibrohistiocytoma. . The malignant tumor is selected from the group consisting of bile duct cancer, prostate cancer, stomach cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, osteosarcoma, Ewing sarcoma and malignant fibrohistiocytoma. 20. The method of claim 19, comprising at least one tumor. The malignant tumor is selected from the group consisting of bile duct cancer, prostate cancer, stomach cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, osteosarcoma, Ewing sarcoma and malignant fibrohistiocytoma. 20. The method of claim 19, comprising all tumors.