JPWO2006006583A1 - Diagnosis of cancer, etc. and identification of primary lesion by detection of HNF4α isoform - Google Patents

Abstract

Provided are a method for determining cancer and a method for specifying the primary tumor site. A protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 in a biological sample, or SEQ ID NOs: 2, 4, 6, 8, 10, 12 , 14, 16 and 18 comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added, and detecting a protein having the function of HNF4α by an immunological technique. A method for specifying a cancer primary lesion that is characterized.

Description

  The present invention relates to a marker HNF4α specific for cancer (gastric cancer, liver cancer, pancreatic cancer, colon cancer, renal cancer, bile duct cancer / gallbladder cancer), pseudobile duct, and intestinal epithelial pseudonym.

  The present inventors have conducted gene expression analysis using DNA chips in normal tissues, cancer cell lines, and various cancer tissues in order to identify targets for diagnosis and treatment of diseases including cancer. As a result, we succeeded in extracting genes with enhanced expression in various cancers. Among them, it was confirmed that the expression of HNF4α mRNA was increased in many cancer tissues.

  HNF4 is an abbreviation for hepatocyte nuclear factor, and there are HNF4α and HNF4β. The gene sequence in rat HNF4α was cloned in 1991 (Patent Document 1), and human HNF4α was cloned in 1994 (Non-Patent Document 1), and the nuclear receptor superfamily involved in metabolic regulation and endoderm development. 1 (Non-Patent Documents 2 and 3), and it was revealed that it is a causative gene of MODY1 (materiality-onset diabetics of the young type 1), but knowledge as a tumor marker was not known. In addition to alternative splicing, human HNF4α has many isoforms due to alternative promoter usage (P1 and P2 promoters), and it is suggested that isoforms with different functions are expressed in different tissues (non-patent literature). 4). HNF4α derived from the P1 promoter is reported to have 1 to 6 and HNF4α1 to 3 are registered for their gene sequences, but HNF4α4 to 6 are not registered. On the other hand, the gene sequence and tissue distribution of human HNF4α derived from the P2 promoter and its function are unknown.

Cancer is on the rise worldwide and is the number one mortality disease in Japan. Among cancers, stomach cancer, liver cancer, pancreatic cancer, colon cancer, renal cancer, bile duct cancer and gallbladder cancer account for a very high ratio. Furthermore, metastasis due to these cancers is frequently seen, and the identification of the primary cancer is an important factor in formulating a treatment policy. In addition, 60% of metastatic cancers of unknown primary origin are adenocarcinomas, and the most common primary lesions are lung cancer and pancreatic cancer, and they generally have a poor prognosis, but they are not rare. (Non-Patent Document 5).
WO9211365 Gene, 1994, Sep. 30; 147 (2): 269-72 J. et al. Mol. Endcrilinol. 2001, 27: 11-29 Endcr. Rev. 1999, 20: 689-725. Mech. Dev. 2001, 109: 183-193. de Almeida PC, Pestana CB: AMB Rev Assoc Med Bras. 1989; 35 (3): 84-7.

  Pathological and radiological searches are used to identify the primary lesion of a cancer of unknown primary origin, and an accurate diagnosis may be reached, but in many cases the primary lesion remains unknown and is finally revealed by pathological anatomy . In this case, it is a method for differentiating by organ, and examination using a specific tumor marker not only consumes a lot of time but also cannot provide sufficient information. Therefore, introduction of a new method to the medical field is expected. It is rare. Against this background, an expression profile analysis of cancer types by microarray analysis is being tried, but its prospect of practical use has not been reached. Therefore, an object of the present invention is to provide a simple and accurate method for determining the primary lesion of a cancer of unknown primary origin.

  HNF4α was known to have 1 to 6 isoforms with high homology, but when the present inventors searched for similar genes, it was different from the conventionally known P1 promoter-derived HNF4α1-6. P2 promoter-derived HNF4α7, HNF4α8 and HNF4α9 have been discovered. Furthermore, it specifically recognizes PNF promoter-derived HNF4α but does not recognize P2 promoter-derived HNF4α, recognizes P2 promoter-derived HNF4α specifically but does not recognize P1 promoter-derived HNF4α, and both Succeeded in producing antibodies. By immunological techniques using these antibodies, it was discovered that the expression of these isoforms varies from tissue to tissue. Based on these results, the present inventors have succeeded in determining the primary lesion of cancer and pseudobile duct, intestinal epithelial pseudonym and cancer of unknown primary, and completed the present invention.

That is, the present invention comprises a protein comprising the amino acid sequence represented by SEQ ID NO: 14, or an amino acid sequence obtained by deleting, substituting, or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 14, and has a function of HNF4α. Provide protein.
The present invention also includes a protein comprising the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 16, and has a function of HNF4α. Provide protein.
The present invention also includes a protein comprising the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 18, and has a function of HNF4α. Provide protein.
The present invention also provides genes encoding the above three proteins.
The present invention also relates to a protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, SEQ ID NOs: 2, 4, 6, 8, 10, 12 A protein having the function of HNF4α consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of any one of, 14, 16 and 18, or the amino acid sequence constituting these proteins The antibody with respect to the peptide which consists of 10-100 amino acids continuous among these is provided.
The present invention also relates to a protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 in a biological sample, or SEQ ID NOs: 2, 4, 6 , 8, 10, 12, 14, 16, and 18 comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and an immunologically functional protein having the function of HNF4α The present invention provides a method for determining cancer, pseudobile duct and intestinal epithelial pseudomorphism characterized by detection by a technique.
Furthermore, the present invention provides a protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 in a biological sample, or SEQ ID NOs: 2, 4, 6, An immunological method comprising a protein having a function of HNF4α, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of any of 8, 10, 12, 14, 16 and 18 It is intended to provide a method for specifying a cancer primary lesion characterized by detecting by the above.

  According to the present invention, diagnosis of many cancers and identification of primary lesions in metastatic cancers can be easily and accurately identified by an immunological technique using an antibody that specifically recognizes PNF and P2 promoter-derived HNF4α. It is possible to provide technology that can answer the needs in the medical field.

It is a figure which shows the reactivity of P1 antibody K9218, P2 antibody H69939, C-terminal antibody H1415, and HNF4 (alpha) isoforms 1-9. The tissue immunostaining image by the antibody of a normal colon is shown. The tissue immunostaining image by the antibody of a normal pancreas is shown. A tissue immunostaining image with an antibody of a normal kidney is shown. The tissue immunostaining image by the antibody of a normal lung is shown. The tissue immunostaining image by the antibody of gastric cancer is shown. The tissue immunostaining image by the antibody of a colon cancer and a thyroid cancer is shown. The tissue immunostaining image by the antibody of pancreatic cancer and liver cancer is shown. The tissue immunostaining image by the antibody of renal cell carcinoma and lung adenocarcinoma is shown.

Hereinafter, the present invention will be described in detail.
The present invention provides a method for diagnosing various cancers and identifying a primary lesion of a cancer of unknown primary by detecting an HNF4α isoform in a biological sample.

  The isoform of HNF4α that is a detection target of the present invention is a protein consisting of the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or SEQ ID NOs: 2, 4, 6, It is a protein having an HNF4α function, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequences described in 8, 10, 12, 14, 16, and 18. Among these, the proteins of SEQ ID NOs: 2, 4, 6, 8, 10, and 12 correspond to HNF4α1 to 6 derived from the P1 promoter, which are known HNF4α isoforms, respectively. On the other hand, the proteins of SEQ ID NOs: 14, 16 and 18 are HNF4α isoforms derived from the P2 promoter newly found by the present inventors. These are referred to as HNF4α7-9, respectively.

  HNF4α7, HNF4α8, and HNF4α9 can be cloned by PCR reaction using primers designed based on the nucleotide sequence of HNF4α1-6 gene from a cDNA library obtained from, for example, a human-derived cancer cell. Examples of cancer cells to be used include Caco-2 and Kato III. As the primer, it is preferable to use the primer of SEQ ID NO: 19 as a forward primer and the primer of SEQ ID NO: 20 or 21 as a reverse primer. The PCR is preferably RT-PCR. Furthermore, the PCR product can be made into a recombinant vector by subcloning into a T vector, fragmenting the cDNA, and inserting it into a known plasmid. By analyzing the gene of the recombinant vector, the HNF4α7, HNF4α8, and HNF4α9 genes can be cloned.

  For expression of the obtained HNF4α7, HNF4α8 or HNF4α9 gene, an expression system using a known expression virus, expression plasmid or the like is used. For example, baculovirus, phage, plasmid or the like is used as an expression vector. In addition, insect cells, yeasts, mammalian cells, E. coli and the like are used as host cells.

  HNF4α1 to HNF4α9 used in the present invention include not only proteins consisting of the amino acid sequences described in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, but also SEQ ID NOs: 2, 4, 6, 8 A protein having a function of HNF4α is also included, comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequences described in 10, 12, 14, 16, and 18. HNF4α1-9 also includes post-translationally modified proteins. There are one or more positions and types to be modified after translation. Post-translational modifications include phosphorylation, saccharification, acetylation, myristoylation and the like. HNF4α1-9 also includes pharmaceutically acceptable salts. The one or several amino acid sequences are deleted, substituted, or amino acid sequence has 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18, More preferably 90% or more is included.

  Further, the gene encoding HNF4α7, HNF4α8 or HNF4α9 includes a base complementary to the gene consisting of the base sequence described in SEQ ID NO: 13, 15 or 17 or the DNA consisting of the base sequence described in SEQ ID NO: 13, 15 or 17. Examples thereof include DNA that hybridizes with a sequence under stringent conditions and encodes a protein having the function of HNF4α. Here, stringent conditions include conditions of 50 ° C. in 0.2 × SSC containing 0.1% SDS and 60 ° C. in 1 × SSC containing 0.1% SDS. DNAs that hybridize under such stringent conditions include those having a homology of 80% or more, more preferably 90% or more, with the base sequence described in SEQ ID NO: 13, 15 or 17.

  The determination method of the present invention determines whether or not the patient is suffering from a disease associated with enhanced expression of any one of HNF4α1 to 9 in a clinical sample. Examples of the disease include cancer (stomach cancer, liver cancer, pancreatic cancer, colon cancer, renal cancer, bile duct cancer / gallbladder cancer), pseudobile duct, and intestinal epithelial pseudonym. The present invention is not limited to the determination only by detecting the protein of the HNF4α isoform, and includes cases where it is used in combination with other diagnostic methods.

  Examples of biological samples used in the method for determining cancer and the method for specifying the primary tumor of the present invention include (a) tissue, (b) collected tissue culture, (c) tissue extract, and (d) cancer patients. And (e) urine or (f) blood. In determination by immunohistochemistry, it is desirable to use (a) to (d). The tissue can be collected by biopsy. The collected tissue can also be used by embedding in paraffin or freezing when immunohistological staining is performed.

  The method for determining cancer and the method for specifying the primary lesion of the present invention are based on an immunological technique using an antibody. Immunological techniques are accomplished by numerous means known to those skilled in the art, including immunohistochemistry, Western blotting, enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay Method (CLIA) and fluorescence immunoassay (FIA).

Specifically, in the case of immunohistochemistry, an anti-HNF4α isoform antibody is detected in a tissue excised by biopsy or surgery, and the HNF4α isoform in the tissue is detected. At the same time, by performing hematoxylin eosin (HE) staining, the relationship between the tissue type and the expression level of the HNF4α isoform can be known.
In the case of Western blotting, proteins obtained from the clinical specimen are separated by polyacrylamide electrophoresis according to the difference in molecular weight, and transferred to a polybilinidene difluoride (PVDF) membrane or a nitrocellulose membrane. Thereafter, anti-HNF4α isoform antibody is allowed to act, and subsequently detection is performed by acting an antibody that recognizes anti-HNF4α isoform antibody labeled with horseradish peroxidase, alkaline phosphatase fluorescent dye, biotin, or the like, that is, a secondary antibody. . Detection may be performed by directly labeling the anti-HNF4α isoform antibody. Confirm that the protein size is HNF4α isoform. This size may be the full length protein of the HNF4α isoform, a post-translationally modified HNF4α isoform, or a fragment of the degraded HNF4α isoform.
In the immunoassay method, any one of HNF4α1 to 9 in a specimen was captured using a solid phase simple substance (for example, immunoplate, latex particle, etc.) to which an antibody was bound by physical adsorption or chemical binding, and then captured. Any of HNF4α1 to 9 can be detected and quantified using a labeled antibody having an antigen recognition site different from the antibody immobilized on the solid phase carrier. It is also possible to solidify a sample on a solid phase carrier, and detect and quantify using a labeled antibody.

  Antibodies are roughly divided into polyclonal antibodies and monoclonal antibodies. Monoclonal antibodies are known to be highly specific because they recognize a single antigenic site. The immunological technique according to the present invention preferably uses a monoclonal antibody. That is, the present invention includes a case where HNF4α1-9 is detected by a monoclonal antibody alone, a plurality of monoclonal antibodies, or a combination of a monoclonal antibody and a polyclonal antibody.

  The antibody used in the present invention may be an endogenous protein or recombinant protein obtained from a living body. Although it is an immunogen for producing an antibody, it may be one or more full lengths of HNF4α protein isoforms or fragments thereof. In addition, a synthetic peptide may be used as an immunogen, but the length thereof is 10 or more consecutive amino acids among the amino acid sequences described in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, 10-100 amino acids, especially 10-80 amino acids are desirable. Examples of such synthetic peptides include peptides of 10 to 100 amino acids on the N-terminal side of HNF4α2 (for example, peptides of the 3rd to 49th amino acids described below), peptides of 10 to 100 amino acids on the C-terminal side of HNF4α8 (for example, those of 380 to 380). 449th amino acid peptide), and a peptide having 10 to 100 amino acids on the N-terminal side of HNF4α8 (for example, a peptide having 1st to 16th amino acids described below).

  The host that produces the recombinant protein is not limited, and examples thereof include viruses, Escherichia coli, yeast, insect cells, mammalian cells, and the like, including production of proteins by a cell-free synthesis system. Recombinant proteins also include fusion proteins. The fusion protein is a fusion protein with glutathione-S-transferase (GST), maltose fusion protein (MBP), thioredoxin, polyhistidine, FLAG sequence, Xpress sequence and the like. Examples of animals that produce antibodies include mammals such as mice, rabbits, goats, sheep, chickens, guinea pigs, and camels. However, animal species are not limited as long as they specifically recognize HNF4α.

  According to the detection results of HNF4α1-9 according to the present invention, cancer, pseudobile duct and intestinal epithelial pseudonym can be determined. For example, an HNF4α isoform derived from the P1 promoter, that is, an antibody that reacts with HNF4α1-6 (P1 antibody), and an HNF4α isoform derived from the P2 promoter, that is, an antibody that reacts with HNF4α7-9 (P2 antibody). It is preferable to detect in combination. Furthermore, it is preferable to detect by combining antibodies (C-terminal antibodies) that react with both part of HNF4α1-6 and part of HNF4α7-9, if necessary. As examples of determination, P1 antibody was positive in almost all cases of liver cancer and gastric cancer, and P2 antibody was positive in 100% of gastric cancer and colon cancer. In addition, almost half of gastric cancer and colorectal cancer were positive for the P1 antibody, and there were also positive cases for biliary tract cancer and gallbladder cancer. Furthermore, P2 antibody was positive in pancreatic cancer. On the other hand, lung cancer, uterine cancer, thyroid cancer, and ovarian cancer were negative for both P1 antibody and P2 antibody.

  Moreover, the primary tumor focus can be specified as shown in Table 3 below, based on the detection results of HNF4α1 to 9 according to the present invention.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this at all.

[Example 1] Cloning of HNF4α7, HNF4α8 and HNF4α9 Total RNA was extracted from several cell types using ISOGEN (Wako Chemical). Specifically, HNF4α7 and 8 were cloned from Caco-2 cells and HNF4α9 was cloned from Kato III cells using RT-PCR using the Expand High Fidelity PCR System. In order to synthesize cDNA, RNA was annealed with a random hexamer, and an extension reaction was performed using SuperScript II reverse transcriptase (Invitrogen). The primers used were SEQ ID NO: 19 for the forward primer, SEQ ID NO: 20 for the reverse primer of HNF4α7 and 8, and SEQ ID NO: 21 for the reverse primer of HNF4α9. The PCR product was subcloned into T vector (Promega), and a fragment obtained by cleaving cDNA with restriction enzyme Not I was inserted into pcDNA3 (Invitrogen). The gene sequence of the produced recombinant was confirmed by the dye terminator method (ABI).

[Example 2] Preparation and culture of peptides expressing amino acids 3 to 49 of HNF4α2 and peptides 380 to 448 of amino acid sequence HNF4α8 (Acquisition of purified virus expression)
The peptide of interest was produced by a recombinant gp64 fusion protein of Autographa californica nuclear polyhedrosis virus (AcMNPV). Regarding the 3rd to 49th amino acid sequences of HNF4α2, SEQ ID NO: 22 was used as a forward primer and SEQ ID NO: 23 was used as a reverse primer. About the amino acid sequence 380th-448th of HNF4 (alpha) 8, sequence number 24 was used as a forward primer and sequence number 25 was used as a reverse primer. Using the human HNF4α8 cDNA as a template, the site corresponding to each peptide was amplified by the PCR method, and a recombinant gene was prepared so as to be a fusion protein of the gp64 gene. The recombinant gene was expressed by inserting the produced recombinant gene into baculovirus DNA. That is, a recombinant virus was prepared by inserting a transfer vector and baculovirus DNA (Bac-N-Blue DNA) (Invitrogen) into Sf9 cells. Subsequently, Graf's Insect Media, 10% fetal bovine serum (FBS) was used for culturing Sf9 cells, and cultured at 27 ° C. for 72 to 120 hours after recombination to produce recombinant virus in the culture supernatant. Virus purification was performed by plaque assay. The plaque assay was performed as follows. 5 × 10 ⁶ cells in a 100 mm dish were cultured in a 10% FBS medium, and a 10-fold diluted virus solution series was infected. After 1 hour, the medium was removed and agar-containing medium was placed. After culturing at 27 ° C. for 6 days and staining with neutral red for 3 hours, plaques were taken with a Pasteur pipette, put into 1 mL of medium, and stirred to elute the virus. The virus was purified by performing this operation twice. 2 × 10 ⁶ Sf9 cells were infected with the purified virus and cultured at 27 ° C. for 10 days. Further, 5 mL of the virus solution was suspended in 500 mL of 2 × 10 ⁶ Sf9 cells / mL suspension culture. After culturing for 10 days, centrifugation was performed at 1000 × g for 20 minutes to obtain a supernatant virus solution. The titer was measured by the plaque assay method, and the virus solution was infected with 500 mL of 2 × 10 ⁶ Sf9 cells / mL suspension culture so that the MOI was 5. Subsequently, in order to express as a gp64 fusion protein, after 3 days of culture, centrifugation was performed at 45000 × g for 30 minutes to obtain a virus in the precipitate fraction, which was then suspended in PBS and purified baculovirus expression product. Got.

[Example 3] Obtaining peptides 1 to 16 of amino acid sequence of HNF4α8 The peptides 1 to 16 of amino acid sequence of HNF4α7 were obtained by consigning synthesis to Peptide Institute.

[Example 4] Preparation of antibody against amino acid sequence 3 to 49 of HNF4α2 and peptide against amino acid sequence 380 to 448 of HNF4α8 Amino acid sequence 3 to 49 of human HNF4α2 and HNF4α8 A purified baculovirus expression product (gp64-fusion expression virus particle) incorporating the amino acid sequence of amino acid sequence 380 to 448 was used as an immunogen.
Mice (BALB / c female 6 weeks old) were immunized 3 times at 100 μg / mouse, and then spleen cells were collected 72 hours later, and myeloma cells (P3 / NSI-1-Ag4-1) and cell fusion (Kohler G Milstein C: Nature 256, 495 (1975)). Hybridomas were obtained by culturing in a HAT selective medium.
Hybridoma culture supernatants were subjected to immune antigen (gp64-expressing virus particle) solid-phase ELISA and wild-type virus particle solid-phase ELISA, and those that react only with the immune antigen were primarily selected.

  The outline of the ELISA method is that an ELISA plate on which 0.5 μg / well of an immunogen and a wild type virus particle is immobilized is reacted with a culture supernatant of a hybridoma, and a substrate is added through a reaction of an HRP-labeled anti-mouse antibody. A method of measuring the absorbance at 450 nm (immunoantigen solid phase ELISA method) for the color development obtained later was used.

  The primary selected vibratorybroma was subjected to western blotting using an immunizing antigen and each wild-type virus particle to perform secondary selection. As for the outline of the Western blot, immunogen and wild-type virus particles were applied at 0.6 μg / lane, and SDS-polyacrylamide gel electrophoresis was performed. Next, it was transferred to a Hybond ECL membrane (manufactured by Amersham Biosciences), and after blocking the transfer membrane, the culture supernatant of the hybridoma was reacted. Next, the chemiluminescence obtained after the reaction with the HRP-labeled anti-mouse antibody and the addition of the ECL detection reagent (Amersham Bioscience) was detected by exposing the X-ray film to light.

  A strain secondarily selected as a strain that reacts only with a band specific to the HNF4α expression product of the immunizing antigen was cloned by a limiting dilution method to establish a monoclonal antibody-producing strain.

  Mouse ascites was obtained by inoculating BALB / c mice with antibody-producing hybridomas. The ascites thus obtained was purified by ammonium sulfate salting out to obtain a monoclonal purified antibody.

[Example 5] Preparation of antibody against peptides 1 to 16 of amino acid sequence of HNF4α8 Conjugate peptides 1 to 16 of amino acid sequence of HNF4α8 to keyhole limpet hemocyanin (hereinafter KLH; manufactured by Calbiochem) This was used as an immunogen.
Mice (BALB / c female 6 weeks old) were immunized 3 times at 100 μg / mouse, and then spleen cells were collected 72 hours later, and myeloma cells (P3 / NSI-1-Ag4-1) and cell fusion (Kohler G Milstein C: Nature 256, 495 (1975)). Hybridomas were obtained by culturing in a HAT selective medium. The hybridoma culture supernatant was subjected to an immunizing antigen peptide solid phase ELISA to select primarily those that react with the immunizing antigen peptide.

  The outline of the ELISA method is to react the hybridoma culture supernatant with an ELISA plate on which 0.5 μg / well of HNF4α8 amino acid sequence 1 to 16 peptide is immobilized, and to react with the HRP-labeled anti-mouse antibody. Then, a method of measuring absorbance at 450 nm (immunoantigen solid phase ELISA method) was used for color development obtained after the addition of the substrate.

  The primary selection of the hybridoma was performed by Western blotting using a cell lysate in which HNF4α8 full-length expression was forcibly expressed in a CHO expression system, and a strain that reacted with full-length HNF4α8 was secondarily selected. Those that were positive for secondary selection were cloned by limiting dilution to establish monoclonal antibody-producing strains. Mouse ascites was obtained by inoculating BALB / c mice with antibody-producing hybridomas. Subsequently, it was purified by ammonium sulfate salting out to obtain a purified monoclonal antibody.

[Example 6] Characteristics of monoclonal antibodies by Western blotting For many of the antibodies obtained, Western blotting using lysates of cells in which the full-length proteins of each isoform of HNF4α1 to 9 were forcibly expressed in the CHO expression system was performed. Going to confirm the ability to recognize the full-length protein and isoform specificity.

  The outline of the Western blot is that CHO cells in which HNF4α8 full-length expression was forcibly expressed and host CHO cell lysate as a negative control were applied at 6 μg / lane, and SDS-polyacrylamide gel electrophoresis was performed. went. Next, the gel was transferred to a Hybond ECL membrane (manufactured by Amersham Biosciences), and after blocking the transfer membrane, the culture supernatant of the hybridoma was reacted. Next, the chemiluminescence obtained after the reaction with the HRP-labeled anti-mouse antibody and the addition of the ECL detection reagent (Amersham Bioscience) was detected by exposing the X-ray film to light.

Cell lysate preparation followed the following procedure.
CHO cells were cultured in a 100 mm dish at 6 × 10 ⁵ , and 18 hours later, TransIT LT-1 transfection reagent (Mirus) and 8 μg of pcDNA3-HNF4α1-9 expression vector were transformed. The transformed HNF4α-expressing cultured cells were cultured for 48 hours, washed twice with ice-cold PBS, and the cells were recovered by scraping. The cells were centrifuged at 2000 × g for 5 minutes, and the cells obtained as a precipitate were suspended in ice-cold lysis buffer. The composition of the buffer was 20 mm Hepes (pH 7.9), 20% (v / v) glycerol, 400 mm Kcl, 0.5 mm EDTA, 1 mm DTT, 2 μg / mL aprotinin, pepstatin A, leupeptin, 0.5 mm PMSF. . The cells were operated with liquid nitrogen and a 37 ° C. freeze saw. This operation was dissolved by repeating 3 minutes 3 times. After dissolution, centrifugation was performed at 20,000 × g for 10 minutes, and the supernatant after centrifugation was measured for protein concentration by the Bradford method (Bio-Rad) to obtain a standard of HNF4α1-9.

  Among the many antibodies obtained, in particular, K9218 as an antibody (P1 antibody) against the peptides 3 to 49 of amino acid sequence of HNF4α2, and H1415 as an antibody (C-terminal antibody) against the peptides of amino acids 380 to 448 of HNF4α8. H69939 was selected as an antibody (P2 antibody) against the peptides 1 to 16 of the amino acid sequence of HNF4α8. That is, P1 antibody K9218 is α1, 2, 3, 4, 5, 6, C-terminal antibody H1415 is α1, 2, 4, 5, 7, 8, and P2 antibody H69939 is α7, 8, 9, respectively. (Fig. 1). A hybridoma producing K9218 (FERM ABP-10363), a hybridoma producing H1415 (FERM ABP-10361), and a hybridoma producing H6939 (FERM ABP-10362) were separately incorporated on September 1, 2004, respectively. Deposited at the Research Institute Patent Biological Depositary Center (Central 1-1, Higashi 1-1-1 Tsukuba, Ibaraki Prefecture 305-8586).

[Example 7] Judgment of cancer (1) Immunostaining After the tissue was adjusted to an appropriate size, formalin was fixed and embedded in paraffin by a method well known to those skilled in the art. In order to suppress non-specific reaction, specimens cut into 4 μm were autoclaved, and after treatment with horse serum, HNF4α was reacted with 1% BSA / PBS at a final concentration of 10 μg / mL for 2 hours at 25 ° C. or 4 ° C. overnight. did. After washing with PBS, the endogenous peroxidase reaction was blocked with methanol containing 0.3% hydrogen peroxide for 30 minutes, and the secondary antibody (MultiPOX, Nichirei, Tokyo) was reacted at room temperature for 1 hour. Finally, it was reacted with 0.1 mg / mL 3,3′-diaminobenzidine (DAB) tetrahydrochloride for 5 minutes, and the nucleus was stained with hematoxylene.

(2) Various cancer tissues Surgery cases and biopsy cases at the Department of Pathology, Niigata University School of Medicine and Pathology Department, Niigata University Medical and Dental Hospital were used. 14 cases of gastric cancer, 18 cases of colon cancer, 3 cases of pancreatic cancer, 6 cases of endometrial cancer, 9 cases of ovarian cancer, 21 cases of lung cancer, 10 cases of thyroid cancer, 2 cases of liver cancer and 25 cases of renal cancer.

(3) Immunostaining in various normal tissues Expression of various HNF4α isoforms in normal tissues was examined.
As a result, the entire HNF4α isoform was expressed in endoderm tissue with the exception of the renal proximal tubule. As shown in Table 1, tissues negative for any of P1, P2, and C-terminal antibodies include tongue, salivary gland, esophagus, bladder, testis, prostate, ovary, uterus, mammary gland, placenta, trachea / bronchi, lung, myocardium Skeletal and smooth muscle, liver (sinus capillary wall cells, Kupffer cells) and kidney (endothelial cells, podocytes, mesangial cells, paraglomerular cells, distal tubules, collecting ducts, Henle Houta. On the other hand, the tissues in which strong positive findings were observed with all antibodies are the duodenum (goblet cells, surface absorption cells,) jejunum / ileum (goblet cells, surface absorption cells, APUD cells) large intestine, and only C-terminal antibody is negative. The liver (hepatocyte) and kidney (proximal tubule), and only the P1 antibody was negative for the pancreas. In ontogeny, it was expressed in yolk sac endoderm cells.

  Examples of tissue immunostaining that showed strong positivity for all antibodies were colon tissue, pancreas for P2 and C-terminal antibody positive examples, kidneys for P1 and P2 positive cases only at one site, and negative examples for both antibodies. This is shown in FIG.

(4) Immunostaining in various tumors The results of examining the expression of HNF4α isoforms in tumors are as follows.

  To summarize the above results, the P1 antibody was positive in almost all cases of liver cancer and renal cancer, and the P2 antibody was 100% positive in gastric cancer and colon cancer. In addition, P1 antibody showed positive findings in almost half of gastric cancer and colorectal cancer. Although the rate of pancreatic cancer was lower than expected, P2 antibody positive findings were obtained, and it was confirmed that there were P1 antibody positive cases even in biliary tract cancer and gallbladder cancer. Lung cancer, uterine cancer, thyroid cancer, and ovarian cancer were all negative.

[Example 8] Specification of primary lesion of cancer of unknown primary From the results of Example 7, it can be estimated with high probability that the following cancer is a primary lesion.

  Representative immunostaining results with the P1, P2 and C-terminal antibodies in the above table are shown in FIGS. Stained images of the tumor are shown below.

  The following shows an example in which the result of the estimation of metastatic cancer performed using the blind was correct by autopsy.

Claims (11)

  A protein comprising the amino acid sequence set forth in SEQ ID NO: 14, or a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 14, and having the function of HNF4α.   A protein comprising the amino acid sequence described in SEQ ID NO: 16, or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 16, and having a function of HNF4α.   A protein comprising the amino acid sequence described in SEQ ID NO: 18, or a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 18, and having the function of HNF4α.   The gene which codes the protein of any one of Claims 1-3.   A protein consisting of the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 A protein having a function of HNF4α, or a continuous 10 of the amino acid sequences constituting these proteins, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in any one of An antibody against a peptide consisting of ˜100 amino acids.   The antibody with respect to the peptide which consists of a continuous 10-100 amino acids among the protein of any one of Claims 1-3, or the amino acid sequence which comprises those proteins.   A protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 in a biological sample, or SEQ ID NOs: 2, 4, 6, 8, 10, 12 , 14, 16 and 18 comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added, and detecting a protein having the function of HNF4α by an immunological technique. Characteristic cancer, pseudobile duct, and intestinal epithelial pseudo method.   A protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18 in a biological sample, or SEQ ID NOs: 2, 4, 6, 8, 10, 12 , 14, 16 and 18 comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added, and detecting a protein having the function of HNF4α by an immunological technique. A method for specifying a cancer primary lesion that is characterized.   The method according to claim 7 or 8, wherein the biological sample is a biological tissue.   The method according to claim 7 or 8, wherein the living tissue is cancer or other diseased tissue.   The object of determination or specification is cancer selected from gastric cancer, liver cancer, pancreatic cancer, colon cancer, renal cancer and bile duct cancer / gallbladder cancer, pseudobile duct and intestinal epithelial pseudo. the method of.