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WO2016002189A1 - Anti-proglucagon antibody - Google Patents

Anti-proglucagon antibody Download PDF

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Publication number
WO2016002189A1
WO2016002189A1 PCT/JP2015/003249 JP2015003249W WO2016002189A1 WO 2016002189 A1 WO2016002189 A1 WO 2016002189A1 JP 2015003249 W JP2015003249 W JP 2015003249W WO 2016002189 A1 WO2016002189 A1 WO 2016002189A1
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Prior art keywords
proglucagon
antibody
amino acid
seq
acid sequence
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French (fr)
Japanese (ja)
Inventor
徹 望月
啓一 大島
慶一 畠山
香菜子 中尾
建 山口
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Shizuoka Prefecture
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Shizuoka Prefecture
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Priority to JP2016531103A priority Critical patent/JP6586648B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to an anti-proglucagon antibody, an anti-proglucagon antibody gene, a proglucagon detection method, and a proglucagon detection kit.
  • Glucagon is a peptide hormone consisting of 29 amino acids generated by proglucagon cleavage (processing) in pancreatic ⁇ cells. Glucagon acts on the liver to convert glycogen into glucose, thereby increasing the blood sugar level. It is known that the amino acid sequence of human pancreatic glucagon matches the amino acid sequence of bovine and porcine pancreatic glucagon (Non-patent Document 1).
  • Proglucagon which is a precursor of glucagon is composed of glycentin-related pancreatic polypeptide (GRPP), glucagon, glucagon-like peptide (GLP) -1, and GLP- in order from the amino (N) terminus. 2 is a protein. Processing of proglucagon differs between the pancreas and the intestinal tract. In the pancreas, mainly GRPP and glucagon are produced, whereas in the intestine, GRPP and glucagon are connected mainly with Glicentin, the C-terminal of glucagon. Oxyntomodulin, GLP-1, and GLP-2, each having 8 amino acid residues, are produced.
  • a tumor marker is a biological factor that increases as cancer progresses. This is one of the clinical laboratory items that are detected using antibodies or the like for factors that are mainly released into the blood. Although it may be used as a screening test for early detection of cancer, it is considered that it is useful at the time of follow-up during treatment and recurrence check because of the effects of false negatives and false positives due to individual differences. .
  • Peptide hormone tumor markers include calcitonin, which is high in medullary thyroid cancer, small cell lung cancer, myeloma, and the like, and gastrin releasing peptide precursor (Progastrin releasing peptide: ProGRP), which is high in small cell lung cancer, lung cancer, Insulin-like growth factor-1 (IGF-1) which shows a high value in prostate cancer is known.
  • ProGRP gastrin releasing peptide precursor
  • IGF-1 Insulin-like growth factor-1
  • Glucagonoma a pancreatic islet alpha cell tumor, is characterized by characteristic skin rash, stomatitis, diabetes, hyperglucagonemia, etc., but its pathological condition is proglucagon, which is a precursor of glucagon and has low biological activity. Increases in the blood (Non-patent Document 2).
  • proglucagon is a candidate substance for a gastric cancer marker (Patent Document 1). It has been found that AZ521, a cultured cell line derived from human gastric cancer, AZ521-P7a, a peritoneal metastasis strain of AZ521, and AZ521-P7a-Assites, a peritoneal metastasis strain, release proglucagon into the culture supernatant.
  • the AZ521 cells were transferred from the Tohoku University Cell Bank to RIKEN Cell Bank (RIKEN registration number RCB2087) as a gastric cancer-derived cell line established in Japan by a Japanese researcher and published in 1989.
  • the cells have also been deposited with the JCRB cell bank (Registered symbol JCRB).
  • JCRB Registered symbol JCRB
  • RIKEN and JCRB Bank announced that the AZ521 cells of RIKEN and JCRB Bank were HuTu80 cells of the duodenal cancer cell line due to the confusion at the time of deposit by the RIKEN BioResource Center on March 26, 2014. (Non-patent Document 3).
  • An object of the present invention is to detect with high sensitivity an anti-proglucagon antibody having high specificity or high detection sensitivity, a gene encoding such an anti-proglucagon antibody, and proglucagon expressed in cells such as duodenal cancer cells. It is in providing the detection method and detection kit which can be performed.
  • the present inventors have selected a peptide that constitutes proglucagon as an antigen for producing an anti-proglucagon antibody in consideration of many years of experience, and produced a polyclonal antibody or a monoclonal antibody using such antigen. An antibody specifically recognizing proglucagon was obtained. In addition, it was confirmed that such antibodies include antibodies with higher detection sensitivity than commercially available anti-proglucagon antibodies. The present invention has been completed based on these findings.
  • the present invention comprises (1) a heavy chain variable region having the amino acid sequence represented by SEQ ID NO: 14 and a light chain variable region having the amino acid sequence represented by SEQ ID NO: 16 Glucagon antibody, (2) a heavy chain variable region having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 16 And an anti-proglucagon antibody characterized by specifically binding to proglucagon, and (3) having the amino acid sequence shown in SEQ ID NOs: 2, 4, and 6
  • a heavy chain variable region comprising a heavy chain complementarity determining region
  • a light chain variable region comprising a light chain complementarity determining region having the amino acid sequences shown in SEQ ID NOs: 8, 10, and 12.
  • an anti-proglucagon antibody characterized by binding to an epitope within amino acid residues 111 to 144 of proglucagon comprising the amino acid sequence shown in SEQ ID NO: 21
  • the anti-proglucagon antibody described in (4) above which binds to an epitope in the 111st to 115th amino acid residues of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21.
  • the present invention also relates to (6) an anti-proglucagon antibody gene characterized by encoding the anti-proglucagon antibody according to any one of (1) to (5), and (7) represented by SEQ ID NO: 13.
  • the anti-proglucagon antibody gene according to (6) above comprising a heavy chain variable region gene having a base sequence and a light chain variable region gene having a base sequence represented by SEQ ID NO: 15.
  • the present invention also relates to (8) a proglucagon detection method characterized by using the anti-proglucagon antibody described in any of (1) to (5) above.
  • the present invention also provides (9) a proglucagon detection kit comprising the anti-proglucagon antibody according to any one of (1) to (5), and (10) the above (9).
  • the present invention relates to a method for diagnosing a tumor that produces proglucagon using the proglucagon detection kit described above.
  • an anti-proglucagon antibody characterized by binding to an epitope in the 61st to 90th amino acid residues of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21, and such Examples thereof include a proglucagon detection method characterized by using an anti-proglucagon antibody, and a proglucagon detection kit comprising such an anti-proglucagon antibody.
  • the anti-proglucagon antibody of the present invention and the anti-proglucagon antibodies of the other embodiments described above are excellent in terms of detection sensitivity and specificity.
  • the anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments have higher detection sensitivity than conventional anti-proglucagon antibodies, such as duodenal cancer that could not be detected by conventional anti-proglucagon antibodies
  • Proglucagon contained in cells can be detected, a method for determining the presence or absence of cancer such as duodenal cancer producing proglucagon, a method for evaluating the recurrence risk of such cancer, and the effectiveness of anticancer agents against the above cancer This method can be advantageously used in the above-described determination methods, screening methods for the above-described cancer suppressors and therapeutic agents, and the like.
  • the anti-proglucagon antibody of the present invention includes an antibody comprising a heavy chain variable region (Fv) having the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16, A heavy chain variable region having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and a light chain having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 16
  • an anti-proglucagon monoclonal antibody can be preferably exemplified, and the isotype of the anti-proglucagon monoclonal antibody is not particularly limited.
  • the heavy chain and light chain constant regions (Fc) in the anti-proglucagon antibody of the present invention and the anti-proglucagon antibodies of the other embodiments described above may be antibodies consisting of immunoglobulin constant regions derived from the same species, Further, it may be a chimeric antibody composed of immunoglobulin constant regions derived from different species.
  • 80% or more identity means that the identity is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, and even more. Preferably, it means 93% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • sequence identity can be calculated using a program commonly used in the art (for example, BLAST, FASTA, etc.).
  • the anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments include monoclonal antibodies, F (ab ′) 2 antibody fragments obtained by digesting monoclonal antibodies with pepsin, F (ab ′) The Fab ′ antibody fragment obtained by reducing two antibody fragments, the antibody fragment such as Fab obtained by digesting a monoclonal antibody with papain, and the heavy chain variable region and the light chain variable region by amino acid crosslinking. Examples include linked scFv (single chain antibody).
  • the anti-proglucagon antibody gene of the present invention is not particularly limited as long as it is an antibody gene encoding the anti-proglucagon antibody of the present invention.
  • a heavy chain variable region gene having the base sequence shown in SEQ ID NO: 13 An antibody gene comprising a light chain variable region gene having the base sequence represented by SEQ ID NO: 15 can be specifically exemplified.
  • the anti-proglucagon antibody of the present invention can be produced as a recombinant antibody by expressing the anti-proglucagon antibody gene by a gene recombination technique.
  • a method for producing a recombinant antibody for example, an anti-proglucagon antibody gene is incorporated into an expression vector, and the expression vector is incorporated into a mammalian cell line such as Chinese hamster ovary (CHO) cell, E. coli, yeast cell, insect cell, plant cell. (PJDelves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P.Shepherd and C.Dean., Monoclonal Antibodies.
  • a chimeric antibody can be prepared based on the technique described in JP-A-2005-245337.
  • the base sequence of the antibody gene incorporated into the expression vector may be optimized for the codon sequence according to the host cell to be expressed.
  • transgenic animals such as mice, cows, goats, sheep, chickens, pigs, etc., into which the anti-proglucagon antibody gene of the present invention has been incorporated, are produced using transgenic animal production technology. Large amounts of antibodies derived from the anti-proglucagon antibody gene can be produced from the inside.
  • a peptide consisting of amino acid residues 111 to 144, preferably 111 to 115, of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21 is converted into a non-human animal such as a mouse or a rat.
  • the anti-proglucagon antibody of the above-mentioned other aspect is the above-mentioned using a peptide consisting of amino acid residues 61 to 90, preferably 71 to 77 of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21. It can obtain by screening by the following method.
  • the anti-proglucagon antibody of the present invention produced by transformed cells, transgenic animals, hybridomas and the like and the anti-proglucagon antibody of the above-mentioned other embodiments are, for example, chromatography using Protein A, Protein G column, ion exchange chromatography, hydrophobic chromatography. Purification using ammonium sulfate salting out method, gel filtration, affinity chromatography or the like.
  • the method for detecting proglucagon according to the present invention and the method for detecting proglucagon according to the other aspect described above may be a method for detecting proglucagon using the anti-proglucagon antibody according to the present invention or the anti-proglucagon antibody according to the other aspect described above.
  • the RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, octalony method using the anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments Western blotting methods, immunohistochemical methods such as immunohistochemistry, and the like.
  • the Western blotting method can be preferably exemplified.
  • Detection of proglucagon using the anti-proglucagon antibody of the present invention or the anti-proglucagon antibody of the above-mentioned other embodiments is performed by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986), Sambrook et al. (MOLECULAR CLONING: ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and the like.
  • the proglucagon detection kit of the present invention and the proglucagon detection kit of the above-mentioned other embodiments are the anti-proglucagon antibodies of the present invention and the above-described other embodiments limited in the use of “to detect proglucagon”. It contains an anti-proglucagon antibody.
  • a kit includes a fluorescent substance, a labeling substance such as horseradish peroxidase (HRP) for detecting the anti-proglucagon antibody of the present invention bound to proglucagon and the anti-proglucagon antibody of the other aspect described above. In general, it includes components such as carriers, pH buffering agents, stabilizers, instruction manuals, and the like, which are generally used in this kind of detection (measurement) kit.
  • HRP horseradish peroxidase
  • the epitope of the antibodies of the two sera was the 12th to 22nd amino acids of proglucagon.
  • the epitopes of the antibodies contained within the range of residues (see FIGS. 4 and 6) and two sera (SCC-MGD-801S and SCC-MGD-802S) residues of amino acids 72 to 78 of proglucagon.
  • the epitopes of the antibodies contained within the group (see FIG. 6) and the two types of sera (SCC-MGD-901S and SCC-MGD-902S) range from amino acid residues 116 to 126 of proglucagon. (See FIGS. 5 and 6). The above results indicate that 6 types of polyclonal antibodies specifically recognizing proglucagon were produced.
  • Proglucagon protein was detected by Western blotting using the above 6 types of anti-proglucagon polyclonal antibodies.
  • the three types of culture supernatant prepared in the above item [Preparation of cell culture supernatant] were subjected to SDS-PAGE (polyacrylamide gel electrophoresis) method to separate proteins by molecular weight on polyacrylamide gel.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • This protein was transferred from a polyacrylamide gel to a PVDF membrane (manufactured by Bio-Rad), blocked with a blocking solution (5% skim milk / TBST), and then reacted with the above 6 types of anti-proglucagon polyclonal antibodies as primary antibodies. .
  • the primary antibody used was the above antiserum diluted 1000 times with TBST. After washing with a TBST solution, a 10,000-fold diluted HRP (horseradish peroxidase) -labeled anti-rabbit antibody was used as a secondary antibody, and detection was carried out with ECL Prime Western Blotting Detection Reagent (manufactured by GE Healthcare) (see FIG. 7). ).
  • ECL Prime Western Blotting Detection Reagent manufactured by GE Healthcare
  • three types of commercially available anti-proglucagon polyclonal antibodies [Anti-Glicentin (Y324), Anti-GLP-1 (Y320), Anti-GLP-2 (Y322), all manufactured by Yanaihara Institute, Ltd.] were used. (See FIG. 8).
  • Proglucagon protein was detected by Western blotting using the culture supernatant of the hybridoma producing the above seven types of anti-proglucagon monoclonal antibodies.
  • the three types of culture supernatant prepared in the above item [Preparation of cell culture supernatant] were subjected to SDS-PAGE (polyacrylamide gel electrophoresis) method to separate proteins by molecular weight on polyacrylamide gel.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • This protein was transferred from a polyacrylamide gel to a PVDF membrane (manufactured by Bio-Rad), blocked with a blocking solution (5% skim milk / TBST), and then reacted with the above seven types of anti-proglucagon monoclonal antibodies as primary antibodies.
  • the primary antibody used was the above antiserum diluted 1000 times with TBST. After washing with TBST solution, an HRP (horseradish peroxidase) -labeled anti-mouse antibody diluted 10,000 times was used as a secondary antibody, and detection was performed by fluorescence detection using ECL Prime Western Blotting Detection Reagent (manufactured by GE Healthcare) (see FIG. 17). ).
  • the epitope of the monoclonal antibody SCC-MGD-913 produced using the proglucagon 111-144 antigen is the 111-115 region and does not contain the glicentin, glucagon, GLP-1 and GLP-2 sequences, the SCC- It was revealed that only proglucagon can be specifically measured by a sandwich method using a combination of MGD-913 and one or two other antibodies.
  • BTQ-101 BTQ-101
  • pCR4-TOPO Invitrogen
  • the base sequence of each cloned DNA fragment was determined with a DNA sequencer using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) (see FIGS. 19 and 20). Further, the heavy chain and light chain complementarity determining regions 1 to 3 (CDR1 to 3) were identified by searching by IGBLAST based on the identified heavy chain and light chain variable region gene sequences (see FIGS. 19 and 20).
  • Heavy chain CDR1 base sequence base sequence represented by SEQ ID NO: 1 heavy chain CDR1 amino acid sequence: amino acid sequence represented by SEQ ID NO: 2 heavy chain CDR2 gene sequence: base sequence heavy chain CDR2 amino acid sequence represented by SEQ ID NO: 3 Amino acid sequence represented by 4 heavy chain CDR3 gene sequence: nucleotide sequence represented by SEQ ID NO: 5 heavy chain CDR3 amino acid sequence: amino acid sequence light chain represented by SEQ ID NO: 6
  • CDR1 gene sequence nucleotide sequence light chain represented by SEQ ID NO: 7
  • CDR1 amino acid sequence amino acid sequence shown in SEQ ID NO: 8 light chain CDR2 gene sequence: nucleotide sequence shown in SEQ ID NO: 9 light chain CDR2 amino acid sequence: amino acid sequence shown in SEQ ID NO: 10 light chain CDR3 gene sequence: SEQ ID NO: 11 Light chain CDR3 amino acid sequence shown: SEQ ID NO: 12 amino acid sequence represented by SEQ ID NO: 5 heavy chain CDR3 gene sequence: amino acid sequence represented by S
  • biotylated proglucagon (61-90) 10 ng / mL was used as the labeled antigen, and proglucagon (61-90) 0-200 ng / mL was used as the standard antigen.
  • biotylated proglucagon (111-144) 10 ng / mL was used as the labeled antigen, and proglucagon (111-144) 0-200 ng / mL was used as the standard antigen.
  • the labeled antigen and the standard antigen are all biotinylated by introducing biotin (manufactured by Sigma-Aldorich) into the peptide prepared in the above [Preparation of antigen] or the peptide prepared in the above [Preparation of antigen]. Peptide.
  • the plate was washed 4 times with PBS, 50 ⁇ L of a luminescent substrate solution (manufactured by R & D) was added to each well, and the mixture was reacted at room temperature in a dark place for 20 minutes, and then the luminescence intensity was measured by ultraviolet absorption at 450 nm. (See FIG. 21).
  • a luminescent substrate solution manufactured by R & D
  • proglucagon contained in the conditioned medium of HutU80 cells was detected using reverse phase HPLC and mass spectrometry.
  • HutU80 cells were cultured in a serum-free medium, and 500 ⁇ L of the obtained culture supernatant was added to an Intrada WP-RP column (250 ⁇ 4.6 mm) (Imtakt).
  • Separate elution of the sample is a linear concentration gradient method in which the mixing ratio of eluent A (0.1% TFA / purified water) and eluent B (0.08% TFA / acetonitrile) is changed to 0-100% in 30 minutes. I went there. The flow rate was 0.75 mL per minute and the eluate was collected every 15 seconds using a 96-well plate.
  • proglucagon was detected mainly in fractions G1-G8.
  • Proglucagon and a protein considered to be the C-terminal part of proglucagon were detected in a small amount in fractions F9-F12 and G9-G12.
  • Glucagon measurement kit see FIG. 24.
  • Glucagon EIA YK090 (manufactured by Yauchihara Institute); it is said that it does not intersect with intestinal glucagon, GLP-1, and GLP-2.
  • Glucagon ELISA EZGGLU-30K (Millipore); does not cross glucagon (1-18) and glucagon (19-29), but 0.5% crosses oxyntomodulin.
  • GLP-1 measurement kit see FIG. 25). 2-1.
  • GLP-1-HS ELISA YK161 (manufactured by Yanaihara Laboratories); the antigen is GLP-1 (7-36) and does not cross glucagon and GLP-2, but GLP-1 (1-36) , And GLP-1 (9-36) are crossed equally, and GLP-1 (1-37) and GLP-1 (7-37) cross about 9%.
  • GLP-1 Total ELISA EZGLP1T-36K (Millipore); antigen is GLP-1 (7-36) and does not cross glucagon, GLP-2, and oxyntomodulin, but GLP-1 (9- 36) is supposed to cross 100%.
  • GLP-2 measurement kit see FIG. 26). 3-1.
  • GLP-2 EIA Human GLP-2 EIA: YK141 (manufactured by Yanaihara Laboratories); the antigen is GLP-2 (1-33), and it is said that it does not cross glucagon and GLP-1.
  • GLP-2 ELISA EZGLP2-37K (manufactured by Millipore); the antigen is GLP-2 (1-33), which is said to cross the GLP-2 (3-33) equally.
  • the reacted immunoreactive elution fraction was different from the main proglucagon elution fraction in both the Yanaihara Institute and Millipore products.
  • the immunologically active elution fraction reacted with the Yauchihara Institute product was different from the main proglucagon elution fraction.
  • the Millipore product a reaction was observed in a wide range of elution fractions including the main proglucagon elution fraction.
  • the immune activity was remarkably low, and the measured value was 0.5% or less as compared with the new proglucagon measuring system prepared above.
  • reaction was observed in main proglucagon elution fractions for both the Yanaihara Laboratory product and the Millipore product. It was 16% or less compared with the new proglucagon measurement system prepared in the above.
  • the new proglucagon measurement system prepared above is superior in specificity and detectability in measuring native proglucagon compared to existing glucagon, GLP-1 and GLP-2 measurement kits. It was shown that.
  • the anti-proglucagon antibody of the present invention and the anti-proglucagon antibodies of the other embodiments described above have higher specificity or higher detection sensitivity than conventional anti-proglucagon antibodies, and thus could not be detected by conventional anti-proglucagon antibodies.
  • Proglucagon contained in cells such as duodenal cancer can be detected, a method for determining the presence or absence of cancer such as duodenal cancer producing proglucagon, a method for evaluating the recurrence risk of such cancer, It can be advantageously used in methods for determining the effectiveness of anticancer agents, screening methods for the above-mentioned cancer inhibitors / therapeutics, etc., and contributes to the development of therapeutic agents for cancers that produce proglucagon.

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Abstract

The present invention addresses the problem of providing an anti-proglucagon antibody which has high specificity or detection sensitivity, a gene encoding the anti-proglucagon antibody, and a detection method and a detection kit with which proglucagon expressed in duodenal carcinoma cells, or the like can be detected with high sensitivity. Provided are: an anti-proglucagon antibody including a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16; an anti-proglucagon antibody comprising a heavy chain variable region that includes a heavy chain complementarity determining region having the amino acid sequence shown in SEQ ID NOs: 2, 4, and 6 and a light chain variable region that includes a light chain complementarity determining region having the amino acid sequence shown in SEQ ID NOs: 8, 10, and 12; an anti-proglucagon antibody which binds to an epitope within amino acid residues 111 to 144 of proglucagon including the amino acid sequence shown in SEQ ID NO: 12; an anti-proglucagon antibody gene encoding the anti-proglucagon antibody; and a method for detecting proglucagon using the anti-proglucagon antibody.

Description

抗プログルカゴン抗体Anti-proglucagon antibody

　本発明は、抗プログルカゴン抗体や、抗プログルカゴン抗体遺伝子や、プログルカゴンの検出方法や、プログルカゴンの検出用キットに関する。 The present invention relates to an anti-proglucagon antibody, an anti-proglucagon antibody gene, a proglucagon detection method, and a proglucagon detection kit.

　グルカゴン（Glucagon）は、膵臓のα細胞においてプログルカゴンの切断（プロセッシング）により生じる、２９アミノ酸から成るペプチドホルモンである。グルカゴンは、肝臓に作用してグリコーゲンをグルコースへと変換し、血糖値を上昇させる働きがある。ヒトの膵グルカゴンのアミノ酸配列は、ウシ及びブタの膵グルカゴンのアミノ酸配列と一致することが知られている（非特許文献１）。 Glucagon is a peptide hormone consisting of 29 amino acids generated by proglucagon cleavage (processing) in pancreatic α cells. Glucagon acts on the liver to convert glycogen into glucose, thereby increasing the blood sugar level. It is known that the amino acid sequence of human pancreatic glucagon matches the amino acid sequence of bovine and porcine pancreatic glucagon (Non-patent Document 1).

　グルカゴンの前駆体であるプログルカゴンは、アミノ（Ｎ）末端から順にグリセンチン関連膵ペプチド（Glicentin-related pancreatic polypeptide：ＧＲＰＰ）、グルカゴン、グルカゴン様ペプチド（Glucagon-like peptide：ＧＬＰ）－１、及びＧＬＰ－２を有するタンパク質である。プログルカゴンのプロセッシングは膵臓と腸管で異なり、膵臓においては、主にＧＲＰＰとグルカゴンが生成されるのに対して、腸管においては、主にＧＲＰＰとグルカゴンが連結したグリセンチン（Glicentin）、グルカゴンのＣ端に８アミノ酸残基を有するオキシントモジュリン（Oxyntomodulin）、ＧＬＰ－１、及びＧＬＰ－２がそれぞれ生成される。 Proglucagon which is a precursor of glucagon is composed of glycentin-related pancreatic polypeptide (GRPP), glucagon, glucagon-like peptide (GLP) -1, and GLP- in order from the amino (N) terminus. 2 is a protein. Processing of proglucagon differs between the pancreas and the intestinal tract. In the pancreas, mainly GRPP and glucagon are produced, whereas in the intestine, GRPP and glucagon are connected mainly with Glicentin, the C-terminal of glucagon. Oxyntomodulin, GLP-1, and GLP-2, each having 8 amino acid residues, are produced.

　腫瘍マーカーとは、癌の進行とともに増加する生体因子のことである。主に血液中に遊離してくる因子に対し、抗体等を使用して検出する臨床検査項目の一つである。癌の早期発見のためのスクリーニング検査として用いられることもあるが、個体差による偽陰性及び偽陽性の影響から、現時点ではむしろ、治療中の経過観察や再発チェックの面において有用と考えられている。ペプチドホルモンの腫瘍マーカーとしては、甲状腺髄様癌、肺小細胞癌、骨髄腫などで高値を示すカルシトニン、肺小細胞癌で高値を示すガストリン放出ペプチド前駆体（Progastrin releasing peptide：ＰｒｏＧＲＰ）、肺癌、前立腺癌で高値を示すインスリン様増殖因子－１（Insulin-like growth factor-1：ＩＧＦ－１）などが知られている。 A tumor marker is a biological factor that increases as cancer progresses. This is one of the clinical laboratory items that are detected using antibodies or the like for factors that are mainly released into the blood. Although it may be used as a screening test for early detection of cancer, it is considered that it is useful at the time of follow-up during treatment and recurrence check because of the effects of false negatives and false positives due to individual differences. . Peptide hormone tumor markers include calcitonin, which is high in medullary thyroid cancer, small cell lung cancer, myeloma, and the like, and gastrin releasing peptide precursor (Progastrin releasing peptide: ProGRP), which is high in small cell lung cancer, lung cancer, Insulin-like growth factor-1 (IGF-1) which shows a high value in prostate cancer is known.

　膵臓ランゲルハンス島α細胞腫瘍であるグルカゴノーマは、特徴的な皮疹、口内炎、糖尿、高グルカゴン血症などを主徴とするが、その病態として、グルカゴンの前駆体である生物学的活性の低いプログルカゴンが血中に増加する（非特許文献２）。 Glucagonoma, a pancreatic islet alpha cell tumor, is characterized by characteristic skin rash, stomatitis, diabetes, hyperglucagonemia, etc., but its pathological condition is proglucagon, which is a precursor of glucagon and has low biological activity. Increases in the blood (Non-patent Document 2).

　本発明者らは先にプログルカゴンが、胃癌マーカーの候補物質であると提案している（特許文献１）。ヒト胃癌由来培養細胞株であるＡＺ５２１、ＡＺ５２１の腹膜転移株であるＡＺ５２１－Ｐ７ａ、腹水転移株であるＡＺ５２１－Ｐ７ａ－Ａｓｃｉｔｅｓが培養上清にプログルカゴンを放出するという知見を得ている。 The present inventors have previously proposed that proglucagon is a candidate substance for a gastric cancer marker (Patent Document 1). It has been found that AZ521, a cultured cell line derived from human gastric cancer, AZ521-P7a, a peritoneal metastasis strain of AZ521, and AZ521-P7a-Assites, a peritoneal metastasis strain, release proglucagon into the culture supernatant.

　前記ＡＺ５２１細胞は、日本人研究者によって日本で樹立され、１９８９年に論文発表されている胃癌由来の細胞株として、東北大細胞バンクから理研細胞バンクに移管を受け（理研登録番号ＲＣＢ２０８７）、また同細胞は、医薬基盤研究所ＪＣＲＢ細胞バンクにも寄託されていた（登録記号ＪＣＲＢ）。しかしながら、２０１４年３月２６日付独立行政法人理化学研究所バイオリソースセンターにより、理研及びＪＣＲＢバンクのＡＺ５２１細胞は、寄託された時点での取り違えにより、十二指腸癌細胞株のＨｕＴu８０細胞であることが発表されている（非特許文献３）。 The AZ521 cells were transferred from the Tohoku University Cell Bank to RIKEN Cell Bank (RIKEN registration number RCB2087) as a gastric cancer-derived cell line established in Japan by a Japanese researcher and published in 1989. The cells have also been deposited with the JCRB cell bank (Registered symbol JCRB). However, RIKEN and JCRB Bank announced that the AZ521 cells of RIKEN and JCRB Bank were HuTu80 cells of the duodenal cancer cell line due to the confusion at the time of deposit by the RIKEN BioResource Center on March 26, 2014. (Non-patent Document 3).

特開２００７－２９２７４６号公報JP 2007-292746 A

Thomsen J. et. al., The amino acid sequence of human glucagon., FEBS Lett. 1972 Apr 1;21(3):315-319.Thomsen J. et. Al., The amino acid sequence of human glucagon., FEBS Lett. 1972 Apr 1; 21 (3): 315-319. 川原田 嘉文 編，「肝・胆・膵の外科―疾患編」:III 膵疾患, 12.グルカゴノーマ，医学図書出版，1994年3月発売Kawaharada, Y., “Surgery of the liver, bile, and pancreas-disease”: III. Pancreatic disease, IV. 12. Glucagonoma, Medical book publication, March 1994. 中村幸夫，ＡＺ５２１細胞について緊急のお知らせ，独立行政法人理化学研究所・細胞材料開発室，平成２６年３月２６日Yukio Nakamura, urgent notice about AZ521 cells, RIKEN Cell Materials Development Office, March 26, 2014

　本発明の課題は、特異性が高い、あるいは検出感度の高い抗プログルカゴン抗体や、かかる抗プログルカゴン抗体をコードする遺伝子や、十二指腸癌細胞等の細胞中に発現するプログルカゴンを高感度で検出できる検出方法及び検出用キットを提供することにある。 An object of the present invention is to detect with high sensitivity an anti-proglucagon antibody having high specificity or high detection sensitivity, a gene encoding such an anti-proglucagon antibody, and proglucagon expressed in cells such as duodenal cancer cells. It is in providing the detection method and detection kit which can be performed.

　本発明者らは、抗プログルカゴン抗体を作製するための抗原として、プログルカゴンを構成するペプチドを長年の経験と勘にたよりながら選択し、かかる抗原を用いてポリクローナル抗体やモノクローナル抗体を作製したところ、プログルカゴンを特異的に認識する抗体が得られた。また、かかる抗体の中には、市販の抗プログルカゴン抗体よりも検出感度の高い抗体が含まれることが確認された。本発明はこれらの知見に基づいて完成するに至ったものである。 The present inventors have selected a peptide that constitutes proglucagon as an antigen for producing an anti-proglucagon antibody in consideration of many years of experience, and produced a polyclonal antibody or a monoclonal antibody using such antigen. An antibody specifically recognizing proglucagon was obtained. In addition, it was confirmed that such antibodies include antibodies with higher detection sensitivity than commercially available anti-proglucagon antibodies. The present invention has been completed based on these findings.

　すなわち、本発明は、（１）配列番号１４に示されるアミノ酸配列を有する重鎖可変領域と、配列番号１６に示されるアミノ酸配列を有する軽鎖可変領域とを備えたことを特徴とする抗プログルカゴン抗体や、（２）配列番号１４に示されるアミノ酸配列と８０％以上の配列同一性のアミノ酸配列を有する重鎖可変領域と、配列番号１６に示されるアミノ酸配列と８０％以上の配列同一性のアミノ酸配列を有する軽鎖可変領域とを備え、プログルカゴンに特異的に結合することを特徴とする抗プログルカゴン抗体や、（３）配列番号２、４、及び６に示されるアミノ酸配列を有する重鎖相補性決定領域を含む重鎖可変領域と、配列番号８、１０、及び１２に示されるアミノ酸配列を有する軽鎖相補性決定領域を含む軽鎖可変領域とを備えたことを特徴とする抗プログルカゴン抗体や、（４）配列番号２１に示されるアミノ酸配列からなるプログルカゴンの１１１～１４４番目のアミノ酸残基内のエピトープに結合することを特徴とする抗プログルカゴン抗体や、（５）配列番号２１に示されるアミノ酸配列からなるプログルカゴンの１１１～１１５番目のアミノ酸残基内のエピトープに結合することを特徴とする上記（４）記載の抗プログルカゴン抗体に関する。 That is, the present invention comprises (1) a heavy chain variable region having the amino acid sequence represented by SEQ ID NO: 14 and a light chain variable region having the amino acid sequence represented by SEQ ID NO: 16 Glucagon antibody, (2) a heavy chain variable region having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 16 And an anti-proglucagon antibody characterized by specifically binding to proglucagon, and (3) having the amino acid sequence shown in SEQ ID NOs: 2, 4, and 6 A heavy chain variable region comprising a heavy chain complementarity determining region, and a light chain variable region comprising a light chain complementarity determining region having the amino acid sequences shown in SEQ ID NOs: 8, 10, and 12. And (4) an anti-proglucagon antibody characterized by binding to an epitope within amino acid residues 111 to 144 of proglucagon comprising the amino acid sequence shown in SEQ ID NO: 21 And (5) the anti-proglucagon antibody described in (4) above, which binds to an epitope in the 111st to 115th amino acid residues of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21.

　また本発明は、（６）上記（１）～（５）のいずれかに記載の抗プログルカゴン抗体をコードすることを特徴とする抗プログルカゴン抗体遺伝子や、（７）配列番号１３に示される塩基配列を有する重鎖可変領域遺伝子と、配列番号１５に示される塩基配列を有する軽鎖可変領域遺伝子とを備えたことを特徴とする上記（６）に記載の抗プログルカゴン抗体遺伝子に関する。 The present invention also relates to (6) an anti-proglucagon antibody gene characterized by encoding the anti-proglucagon antibody according to any one of (1) to (5), and (7) represented by SEQ ID NO: 13. The anti-proglucagon antibody gene according to (6) above, comprising a heavy chain variable region gene having a base sequence and a light chain variable region gene having a base sequence represented by SEQ ID NO: 15.

　また本発明は、（８）上記（１）～（５）のいずれかに記載の抗プログルカゴン抗体を用いることを特徴とするプログルカゴンの検出方法に関する。 The present invention also relates to (8) a proglucagon detection method characterized by using the anti-proglucagon antibody described in any of (1) to (5) above.

　また本発明は、（９）上記（１）～（５）のいずれかに記載の抗プログルカゴン抗体を備えたことを特徴とするプログルカゴンの検出用キットや、（１０）上記（９）に記載のプログルカゴンの検出用キットを用いた、プログルカゴンを産生する腫瘍の診断方法に関する。 The present invention also provides (9) a proglucagon detection kit comprising the anti-proglucagon antibody according to any one of (1) to (5), and (10) the above (9). The present invention relates to a method for diagnosing a tumor that produces proglucagon using the proglucagon detection kit described above.

　また本発明の実施の他の形態として、配列番号２１に示されるアミノ酸配列からなるプログルカゴンの６１～９０番目のアミノ酸残基内のエピトープに結合することを特徴とする抗プログルカゴン抗体や、かかる抗プログルカゴン抗体を用いることを特徴とするプログルカゴンの検出方法や、かかる抗プログルカゴン抗体を備えたことを特徴とするプログルカゴンの検出用キットを挙げることができる。 As another embodiment of the present invention, an anti-proglucagon antibody characterized by binding to an epitope in the 61st to 90th amino acid residues of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21, and such Examples thereof include a proglucagon detection method characterized by using an anti-proglucagon antibody, and a proglucagon detection kit comprising such an anti-proglucagon antibody.

　本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体は、検出感度及び特異性の面で優れている。また、本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体は、従来の抗プログルカゴン抗体よりも検出感度が高いため、従来の抗プログルカゴン抗体では検出できなかった十二指腸癌等の細胞中に含まれるプログルカゴンを検出することができ、プログルカゴンを産生する十二指腸癌等の癌の有無を判定する方法や、かかる癌の再発リスクを評価する方法や、上記癌に対する抗癌剤の有効性の判定方法や、上記癌の抑制剤・治療剤のスクリーニング方法等に有利に用いることができる。 The anti-proglucagon antibody of the present invention and the anti-proglucagon antibodies of the other embodiments described above are excellent in terms of detection sensitivity and specificity. In addition, since the anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments have higher detection sensitivity than conventional anti-proglucagon antibodies, such as duodenal cancer that could not be detected by conventional anti-proglucagon antibodies Proglucagon contained in cells can be detected, a method for determining the presence or absence of cancer such as duodenal cancer producing proglucagon, a method for evaluating the recurrence risk of such cancer, and the effectiveness of anticancer agents against the above cancer This method can be advantageously used in the above-described determination methods, screening methods for the above-described cancer suppressors and therapeutic agents, and the like.

プログルカゴン特異抗体の作製における抗原デザインを示す図である。It is a figure which shows the antigen design in preparation of a proglucagon specific antibody. ＡＺ５２１無血清培養上清のＨＰＬＣ各フラクション中のプログルカゴンに対するポリクローナル抗体の免疫反応性を示す図である。It is a figure which shows the immunoreactivity of the polyclonal antibody with respect to the proglucagon in each HPLC fraction of the AZ521 serum-free culture supernatant. ポリクローナル抗体のエピトープ解析に用いたペプチド群を示す図である。It is a figure which shows the peptide group used for the epitope analysis of a polyclonal antibody. ポリクローナル抗体のプログルカゴン２－３０抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to proglucagon 2-30 antigen of a polyclonal antibody. ポリクローナル抗体のプログルカゴン１１１－１４４抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescent immunoassay with respect to the proglucagon 111-144 antigen of a polyclonal antibody. ポリクローナル抗体のエピトープ解析の結果をまとめた図である。It is the figure which put together the result of the epitope analysis of a polyclonal antibody. プログルカゴンを含む各腫瘍細胞株の無血清培養上清サンプルと、作製したポリクローナル抗体によるウェスタンブロッティング解析の結果を示す図である。It is a figure which shows the result of the western blotting analysis by the serum-free culture supernatant sample of each tumor cell line containing proglucagon, and the produced polyclonal antibody. プログルカゴンを含む各腫瘍細胞株の無血清培養上清サンプルと、市販のポリクローナル抗体（抗血清）によるウェスタンブロッティング解析の結果を示す図である。It is a figure which shows the result of the western blotting analysis by the serum-free culture supernatant sample of each tumor cell line containing proglucagon, and a commercially available polyclonal antibody (antiserum). モノクローナル抗体のエピトープ解析に用いたペプチド群を示す図である。It is a figure which shows the peptide group used for the epitope analysis of a monoclonal antibody. モノクローナル抗体８１１Ａｂのプログルカゴン６１－９０抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 61-90 antigen of the monoclonal antibody 811Ab. モノクローナル抗体８１２Ａｂのプログルカゴン６１－９０抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 61-90 antigen of the monoclonal antibody 812Ab. モノクローナル抗体８１３Ａｂのプログルカゴン６１－９０抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 61-90 antigen of the monoclonal antibody 813Ab. モノクローナル抗体９１１Ａｂのプログルカゴン１１１－１４４抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 111-144 antigen of monoclonal antibody 911 Ab. モノクローナル抗体９１２Ａｂのプログルカゴン１１１－１４４抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 111-144 antigen of monoclonal antibody 912Ab. モノクローナル抗体９１３Ａｂのプログルカゴン１１１－１４４抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 111-144 antigen of monoclonal antibody 913Ab. モノクローナル抗体９１４Ａｂのプログルカゴン１１１－１４４抗原に対する、化学発光免疫測定法によるエピトープ解析の結果を示す図である。It is a figure which shows the result of the epitope analysis by the chemiluminescence immunoassay method with respect to the proglucagon 111-144 antigen of monoclonal antibody 914Ab. プログルカゴンを含む各腫瘍細胞株の無血清培養上清サンプルと、作製したモノクローナル抗体によるウェスタンブロッティング解析の結果を示す図である。It is a figure which shows the result of the Western blotting analysis by the serum-free culture supernatant sample of each tumor cell line containing proglucagon, and the produced monoclonal antibody. ９１３Ａｂに対するシークエンス解析のプライマー設定を示す図である。It is a figure which shows the primer setting of the sequence analysis with respect to 913Ab. 抗プログルカゴン抗体（９１３Ａｂ）重鎖のアミノ酸及び塩基配列を示す図である。It is a figure which shows the amino acid and base sequence of an anti-proglucagon antibody (913Ab) heavy chain. 抗プログルカゴン抗体（９１３Ａｂ）軽鎖のアミノ酸及び塩基配列を示す図である。It is a figure which shows the amino acid and base sequence of an anti-proglucagon antibody (913Ab) light chain. ポリクローナル抗体７０２Ｓ、モノクローナル抗体８１２Ａｂ、及びモノクローナル抗体９１３Ａｂを用いたプログルカゴン測定系について、市販品のグルカゴン、ＧＬＰ－１、及びＧＬＰ－２との交差反応性を検討した結果を示す図である。It is a figure which shows the result of having investigated the cross-reactivity with the commercially available glucagon, GLP-1, and GLP-2 about the proglucagon measuring system using the polyclonal antibody 702S, the monoclonal antibody 812Ab, and the monoclonal antibody 913Ab. ＨｕｔＵ８０無血清培養上清のＨＰＬＣ各フラクション中に含まれるタンパク質を質量分析計を用いて分析し、各タンパク質がどのフラクションに溶出されているかを示した図である。It is the figure which analyzed which protein contained in each HPLC fraction of HutU80 serum-free culture supernatant using a mass spectrometer, and which fraction was eluted with each protein. 図２２にて集めた各分画中のタンパク質を、新規プログルカゴン測定系を用いて測定した結果を示す図である。It is a figure which shows the result of having measured the protein in each fraction collected in FIG. 22 using the novel proglucagon measuring system. 図２２にて集めた各分画中のタンパク質を、市販のグルカゴン測定キットを用いて測定した結果を示す図である。It is a figure which shows the result of having measured the protein in each fraction collected in FIG. 22 using a commercially available glucagon measuring kit. 図２２にて集めた各分画中のタンパク質を、市販のＧＬＰ－１測定キットを用いて測定した結果を示す図である。It is a figure which shows the result of having measured the protein in each fraction collected in FIG. 22 using a commercially available GLP-1 measuring kit. 図２２にて集めた各分画中のタンパク質を、市販のＧＬＰ－２測定キットを用いて測定した結果を示す図である。It is a figure which shows the result of having measured the protein in each fraction collected in FIG. 22 using a commercially available GLP-2 measuring kit.

　本発明の抗プログルカゴン抗体としては、配列番号１４に示されるアミノ酸配列を有する重鎖可変領域（Ｆｖ）と、配列番号１６に示されるアミノ酸配列を有する軽鎖可変領域とを備えた抗体や、配列番号１４に示されるアミノ酸配列と８０％以上の配列同一性のアミノ酸配列を有する重鎖可変領域と、配列番号１６に示されるアミノ酸配列と８０％以上の配列同一性のアミノ酸配列を有する軽鎖可変領域とを備え、プログルカゴンに特異的に結合する抗体や、配列番号２、４、及び６に示されるアミノ酸配列を有する重鎖相補性決定領域（complementarity-determining region；ＣＤＲ）（それぞれ重鎖ＣＤＲ１～３）を含む重鎖可変領域と、配列番号８、１０、及び１２に示されるアミノ酸配列を有する軽鎖ＣＤＲ（それぞれ軽鎖ＣＤＲ１～３）を含む軽鎖可変領域とを備えた抗体や、配列番号２１に示されるアミノ酸配列からなるプログルカゴンの１１１～１４４番目のアミノ酸残基内のエピトープに結合する抗体であれば特に制限されない。 The anti-proglucagon antibody of the present invention includes an antibody comprising a heavy chain variable region (Fv) having the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16, A heavy chain variable region having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and a light chain having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 16 An antibody that specifically binds to proglucagon and a heavy chain complementarity-determining region (CDR) having the amino acid sequences shown in SEQ ID NOs: 2, 4, and 6 (each heavy chain) A heavy chain variable region comprising CDR1-3) and a light chain CDR having the amino acid sequence shown in SEQ ID NOs: 8, 10, and 12 (light chain CDR1-3, respectively) Antibodies and that includes a light chain variable region comprising not particularly limited as long as an antibody that binds to an epitope of 111 to 144 amino acid residues of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21.

　本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体としては、抗プログルカゴンモノクローナル抗体を好適に例示することができ、ここで抗プログルカゴンモノクローナル抗体のアイソタイプは特に制限されない。 As the anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments, an anti-proglucagon monoclonal antibody can be preferably exemplified, and the isotype of the anti-proglucagon monoclonal antibody is not particularly limited.

　本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体における重鎖及び軽鎖の定常領域（Ｆｃ）としては、同じ生物種由来の免疫グロブリン定常領域からなる抗体であってもよく、また、異なる生物種由来の免疫グロブリン定常領域からなるキメラ抗体であってもよい。 The heavy chain and light chain constant regions (Fc) in the anti-proglucagon antibody of the present invention and the anti-proglucagon antibodies of the other embodiments described above may be antibodies consisting of immunoglobulin constant regions derived from the same species, Further, it may be a chimeric antibody composed of immunoglobulin constant regions derived from different species.

　本発明において、「８０％以上の同一性」とは、同一性が８０％以上であることを意味し、好ましくは８５％以上、より好ましくは８８％以上、さらに好ましくは９０％以上、さらにより好ましくは９３％以上、特に好ましくは９５％以上、最も好ましくは９８％以上の同一性を意味する。配列同一性は、当該分野で慣用のプログラム（例えば、ＢＬＡＳＴ、ＦＡＳＴＡ等）を用いて算出することができる。 In the present invention, “80% or more identity” means that the identity is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, and even more. Preferably, it means 93% or more, particularly preferably 95% or more, and most preferably 98% or more. The sequence identity can be calculated using a program commonly used in the art (for example, BLAST, FASTA, etc.).

　本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体の種類としては、モノクローナル抗体の他、モノクローナル抗体をペプシンで消化して得られるＦ（ａｂ′）２抗体フラグメントや、Ｆ（ａｂ′）２抗体フラグメントを還元して得られるＦａｂ′抗体フラグメントや、モノクローナル抗体をパパインで消化して得られるＦａｂ等の抗体フラグメントや、上記重鎖可変領域と軽鎖可変領域とを、アミノ酸架橋によって連結させたｓｃＦｖ（１本鎖抗体）等を挙げることができる。 The anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments include monoclonal antibodies, F (ab ′) 2 antibody fragments obtained by digesting monoclonal antibodies with pepsin, F (ab ′) The Fab ′ antibody fragment obtained by reducing two antibody fragments, the antibody fragment such as Fab obtained by digesting a monoclonal antibody with papain, and the heavy chain variable region and the light chain variable region by amino acid crosslinking. Examples include linked scFv (single chain antibody).

　本発明の抗プログルカゴン抗体遺伝子としては、上記本発明の抗プログルカゴン抗体をコードする抗体遺伝子であれば特に制限されず、例えば、配列番号１３に示される塩基配列を有する重鎖可変領域遺伝子と、配列番号１５に示される塩基配列を有する軽鎖可変領域遺伝子とを備えた抗体遺伝子を具体的に例示することができる。 The anti-proglucagon antibody gene of the present invention is not particularly limited as long as it is an antibody gene encoding the anti-proglucagon antibody of the present invention. For example, a heavy chain variable region gene having the base sequence shown in SEQ ID NO: 13 An antibody gene comprising a light chain variable region gene having the base sequence represented by SEQ ID NO: 15 can be specifically exemplified.

　本発明の抗プログルカゴン抗体は、遺伝子組換え技術により、上記抗プログルカゴン抗体遺伝子を発現させることにより、組換え抗体として作製することができる。組換え抗体を作製する方法としては、例えば抗プログルカゴン抗体遺伝子を発現ベクターに組み込み、かかる発現ベクターをチャイニーズハムスター卵巣（ＣＨＯ）細胞等の哺乳類細胞株や、大腸菌、酵母細胞、昆虫細胞、植物細胞等の宿主細胞へ導入して、宿主細胞において組換え抗体を生産させる方法を挙げることができる（P.J.Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY、P.Shepherd and C.Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS, J.W.Goding., Monoclonal Antibodies:principles and practice., 1993 ACADEMIC PRESS）。特に、キメラ抗体は、特開２００５－２４５３３７に記載の技術に基づいて作製することができる。発現ベクターに組み込む抗体遺伝子の塩基配列は、発現させる宿主細胞に合わせてコドン配列の最適化がされていてもよい。 The anti-proglucagon antibody of the present invention can be produced as a recombinant antibody by expressing the anti-proglucagon antibody gene by a gene recombination technique. As a method for producing a recombinant antibody, for example, an anti-proglucagon antibody gene is incorporated into an expression vector, and the expression vector is incorporated into a mammalian cell line such as Chinese hamster ovary (CHO) cell, E. coli, yeast cell, insect cell, plant cell. (PJDelves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P.Shepherd and C.Dean., Monoclonal Antibodies. 2000 OXFORD UNIVERSITY PRESS, JWGoding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS). In particular, a chimeric antibody can be prepared based on the technique described in JP-A-2005-245337. The base sequence of the antibody gene incorporated into the expression vector may be optimized for the codon sequence according to the host cell to be expressed.

　また、トランスジェニック動物作製技術を用いて本発明の抗プログルカゴン抗体遺伝子が組み込まれたマウス、ウシ、ヤギ、ヒツジ、ニワトリ、ブタ等のトランスジェニック動物を作製し、かかるトランスジェニック動物の血液、ミルク中などから上記抗プログルカゴン抗体遺伝子に由来する抗体を大量に産生させることもできる。 In addition, transgenic animals such as mice, cows, goats, sheep, chickens, pigs, etc., into which the anti-proglucagon antibody gene of the present invention has been incorporated, are produced using transgenic animal production technology. Large amounts of antibodies derived from the anti-proglucagon antibody gene can be produced from the inside.

　さらに、慣用のプロトコールを用いて、配列番号２１に示されるアミノ酸配列からなるプログルカゴンの１１１～１４４番目、好ましくは１１１～１１５番目のアミノ酸残基からなるペプチドをマウス、ラット等のヒト以外の動物へ投与し、抗プログルカゴン抗体を産生する細胞クローンを細胞融合技術（ハイブリドーマ法［Nature 256, 495-497, 1975］、トリオーマ法、ヒトＢ細胞ハイブリドーマ法［Immunology Today 4, 72, 1983］及びＥＢＶ－ハイブリドーマ法［MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R.Liss, Inc.,1985］等）によりスクリーニングすることにより、本発明の抗プログルカゴン抗体を得ることができる。また、上記他の態様の抗プログルカゴン抗体については、配列番号２１に示されるアミノ酸配列からなるプログルカゴンの６１～９０番目、好ましくは７１～７７番目のアミノ酸残基からなるペプチドを用いて上述のとおりの方法でスクリーニングすることにより得ることができる。 Further, using a conventional protocol, a peptide consisting of amino acid residues 111 to 144, preferably 111 to 115, of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21 is converted into a non-human animal such as a mouse or a rat. Cell clones that produce anti-proglucagon antibodies by cell fusion technology (hybridoma method [Nature 256, 495-497, 1975], trioma method, human B cell hybridoma method [Immunology Today 4, 72, 1983] and EBV -The anti-proglucagon antibody of the present invention can be obtained by screening by the hybridoma method [MONOCLONAL ANTIBODIES AND CANCER THERAPY, 77pp.77-96, Alan R.Liss, Inc., 1985]. The anti-proglucagon antibody of the above-mentioned other aspect is the above-mentioned using a peptide consisting of amino acid residues 61 to 90, preferably 71 to 77 of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21. It can obtain by screening by the following method.

　形質転換細胞、トランスジェニック動物、ハイブリドーマ等により産生された本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体は、例えばＰｒｏｔｅｉｎＡ、ＰｒｏｔｅｉｎＧカラムによるクロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、硫安塩析法、ゲル濾過、アフィニティクロマトグラフィー等を用いて精製することができる。 The anti-proglucagon antibody of the present invention produced by transformed cells, transgenic animals, hybridomas and the like and the anti-proglucagon antibody of the above-mentioned other embodiments are, for example, chromatography using Protein A, Protein G column, ion exchange chromatography, hydrophobic chromatography. Purification using ammonium sulfate salting out method, gel filtration, affinity chromatography or the like.

　本発明のプログルカゴンの検出方法や上記他の態様のプログルカゴンの検出方法としては、本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体を用いてプログルカゴンを検出する方法であればよく、具体的には本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体を用いたＲＩＡ法、ＥＬＩＳＡ法、蛍光抗体法、プラーク法、スポット法、血球凝集反応法、オクタロニー法、ウェスタンブロッティング法、免疫組織化学等の免疫学的測定方法を挙げることができ、これらの中でもウェスタンブロッティング法を好適に例示することができる。本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体を用いたプログルカゴンの検出は、Davisら（BASIC METHODS IN MOLECULAR BIOLOGY, 1986）、Sambrookら（MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989）などの標準的な実験室マニュアルに記載される方法等により行うことができる。 The method for detecting proglucagon according to the present invention and the method for detecting proglucagon according to the other aspect described above may be a method for detecting proglucagon using the anti-proglucagon antibody according to the present invention or the anti-proglucagon antibody according to the other aspect described above. Specifically, the RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, octalony method using the anti-proglucagon antibody of the present invention and the anti-proglucagon antibody of the above-mentioned other embodiments , Western blotting methods, immunohistochemical methods such as immunohistochemistry, and the like. Among these, the Western blotting method can be preferably exemplified. Detection of proglucagon using the anti-proglucagon antibody of the present invention or the anti-proglucagon antibody of the above-mentioned other embodiments is performed by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986), Sambrook et al. (MOLECULAR CLONING: ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and the like.

　本発明のプログルカゴンの検出用キットや上記他の態様のプログルカゴンの検出用キットは、「プログルカゴンを検出するため」という用途が限定された本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体を含むものである。かかるキットには、プログルカゴンに結合した本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体を検出するための、蛍光物質、西洋ワサビペルオキシダーゼ(Horse Radish Peroxidase;ＨＲＰ)等の標識物質をコンジュゲートした抗体（２次抗体）や、一般にこの種の検出（測定）キットに用いられる成分、例えば担体、ｐＨ緩衝剤、安定剤、取扱説明書等の添付文書が通常含まれる。 The proglucagon detection kit of the present invention and the proglucagon detection kit of the above-mentioned other embodiments are the anti-proglucagon antibodies of the present invention and the above-described other embodiments limited in the use of “to detect proglucagon”. It contains an anti-proglucagon antibody. Such a kit includes a fluorescent substance, a labeling substance such as horseradish peroxidase (HRP) for detecting the anti-proglucagon antibody of the present invention bound to proglucagon and the anti-proglucagon antibody of the other aspect described above. In general, it includes components such as carriers, pH buffering agents, stabilizers, instruction manuals, and the like, which are generally used in this kind of detection (measurement) kit.

　以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

［細胞の培養上清の調製］
　十二指腸癌由来の３種類の細胞株（ＡＺ－５２１、ＡＺ５２１－Ｐ７ａ、及びＨｕＴu８０）１×１０６個を１０ｃｍシャーレに播種し、１０％のウシ胎児血清（fetal bovine serum：ＦＢＳ）を含むＲＰＭＩ１６４０培養液（Sigma社製）存在下で培養し、２４時間後にリン酸緩衝生理食塩水（phosphate buffered saline：ＰＢＳ）で２回洗浄した後、ＦＢＳを含まないＲＰＭＩ１６４０培養液に交換し、１時間培養した後、培養液を新しいＲＰＭＩ１６４０培養液に置換し、さらに４８時間培養した。４８時間後に培養液を回収し、本実施例における培養上清として用いた。なお、細胞培養は、３７℃、５％ＣＯ２条件下で行った。 [Preparation of cell culture supernatant]
RPMI1640 culture containing 10% fetal bovine serum (FBS) after seeding 1 × 10 6 cell lines (AZ-521, AZ521-P7a, and HuTu80) derived from 3 types of duodenal cancer After culturing in the presence of a liquid (Sigma), washed with phosphate buffered saline (PBS) 24 hours later, the medium was replaced with RPMI 1640 medium containing no FBS and cultured for 1 hour. Thereafter, the culture solution was replaced with a new RPMI 1640 culture solution, and the culture was further continued for 48 hours. After 48 hours, the culture solution was collected and used as the culture supernatant in this example. The cell culture was performed under conditions of 37 ° C. and 5% CO 2 .

［抗原の作製］
　図１に示す３種類の抗原のアミノ酸配列に相当するペプチドを化学合成した。化学合成は、自動ペプチド合成機（430A、Applide biosystems社製）を用い、合成機搭載のプログラムによりＦ－ｍｏｃ－アミノ酸を端より順次結合して行った。合成ペプチドの精製は分取用逆相ＨＰＬＣにより行い、純度は分析用逆相ＨＰＬＣで確認した。合成品の分子量は質量分析により理論値に一致することを確認した。 [Preparation of antigen]
Peptides corresponding to the amino acid sequences of the three antigens shown in FIG. 1 were chemically synthesized. Chemical synthesis was performed using an automatic peptide synthesizer (430A, manufactured by Applide biosystems), and sequentially connecting F-moc-amino acids from the end by a program installed on the synthesizer. The synthetic peptide was purified by preparative reverse phase HPLC, and the purity was confirmed by analytical reverse phase HPLC. The molecular weight of the synthesized product was confirmed by mass spectrometry to agree with the theoretical value.

［ポリクローナル抗体の作製］
　上記３種類のプログルカゴンペプチド（１ｍｇ）それぞれをキーホールリンペットヘモシアニン（KLH、５ｍｇ）と結合したコンジュゲートを、日本白色種ウサギ２匹に週１回、全６回注射して免疫した後、最終の免疫から１週間後にその全血を採取し、血清を全回収し、保存料として０．０９％のアジ化ナトリウムを加えた。すなわち、プログルカゴンの２～３０番目のアミノ酸残基からなるペプチドを免疫して得られた血清２種（ＳＣＣ－ＭＧＤ－７０１Ｓ及びＳＣＣ－ＭＧＤ－７０２Ｓ）、プログルカゴンの６１～９０番目のアミノ酸残基からなるペプチドを免疫して得られた血清２種（ＳＣＣ－ＭＧＤ－８０１Ｓ及びＳＣＣ－ＭＧＤ－８０２Ｓ）、及びプログルカゴンの１１１～１４４番目のアミノ酸残基からなるペプチドを免疫して得られた血清２種（ＳＣＣ－ＭＧＤ－９０１Ｓ及びＳＣＣ－ＭＧＤ－９０２Ｓ）の計６種の血清が得られた。 [Preparation of polyclonal antibody]
After conjugating two Japanese white rabbits with a conjugate of each of the three types of proglucagon peptides (1 mg) and keyhole limpet hemocyanin (KLH, 5 mg) once a week for a total of 6 injections, One week after the final immunization, the whole blood was collected, the whole serum was collected, and 0.09% sodium azide was added as a preservative. That is, two kinds of sera obtained by immunization with peptides consisting of the 2-30th amino acid residues of proglucagon (SCC-MGD-701S and SCC-MGD-702S), the 61st-90th amino acid residues of proglucagon Two sera obtained by immunizing a peptide comprising a group (SCC-MGD-801S and SCC-MGD-802S) and obtained by immunizing a peptide consisting of amino acid residues 111 to 144 of proglucagon A total of 6 sera, 2 sera (SCC-MGD-901S and SCC-MGD-902S), were obtained.

［ポリクローナル抗体の免疫反応性とエピトープの検定］
　抗体の免疫反応性を検定するために、４種類の血清（ＳＣＣ－ＭＧＤ－７０１Ｓ及びＳＣＣ－ＭＧＤ－７０２Ｓ、並びにＳＣＣ－ＭＧＤ－８０１Ｓ及びＳＣＣ－ＭＧＤ－８０２Ｓ）を用いてＨＰＬＣで分画したＡＺ－５２１細胞株の培養上清画分と反応させた（図２参照）。その結果、プログルカゴンが特に多く含まれる画分（Ｆ１０－１２）に対する免疫反応性が高いことが示された。
　また、抗体のエピトープについて詳細に解析したところ（図３～６参照）、血清２種（ＳＣＣ－ＭＧＤ－７０１Ｓ及びＳＣＣ－ＭＧＤ－７０２Ｓ）の抗体のエピトープは、プログルカゴンの１２～２２番目のアミノ酸残基の範囲内に含まれることや（図４、６参照）、血清２種（ＳＣＣ－ＭＧＤ－８０１Ｓ及びＳＣＣ－ＭＧＤ－８０２Ｓ）の抗体のエピトープは、プログルカゴンの７２～７８番目のアミノ酸残基の範囲内に含まれることや（図６参照）、血清２種（ＳＣＣ－ＭＧＤ－９０１Ｓ及びＳＣＣ－ＭＧＤ－９０２Ｓ）の抗体のエピトープは、プログルカゴンの１１６～１２６番目のアミノ酸残基の範囲内に含まれることが明らかとなった（図５、６参照）。
　以上の結果は、プログルカゴンを特異的に認識するポリクローナル抗体６種が作製されたことを示している。 [Immunoreactivity of polyclonal antibodies and epitope assay]
AZ fractionated by HPLC using 4 types of sera (SCC-MGD-701S and SCC-MGD-702S, and SCC-MGD-801S and SCC-MGD-802S) to assay antibody immunoreactivity It was reacted with the culture supernatant fraction of the −521 cell line (see FIG. 2). As a result, it was shown that the immunoreactivity with a fraction (F10-12) containing a particularly large amount of proglucagon was high.
Further, when the epitope of the antibody was analyzed in detail (see FIGS. 3 to 6), the epitope of the antibodies of the two sera (SCC-MGD-701S and SCC-MGD-702S) was the 12th to 22nd amino acids of proglucagon. The epitopes of the antibodies contained within the range of residues (see FIGS. 4 and 6) and two sera (SCC-MGD-801S and SCC-MGD-802S) residues of amino acids 72 to 78 of proglucagon The epitopes of the antibodies contained within the group (see FIG. 6) and the two types of sera (SCC-MGD-901S and SCC-MGD-902S) range from amino acid residues 116 to 126 of proglucagon. (See FIGS. 5 and 6).
The above results indicate that 6 types of polyclonal antibodies specifically recognizing proglucagon were produced.

［ポリクローナル抗体を用いたウェスタンブロッティング法］
　上記６種類の抗プログルカゴンポリクローナル抗体を用いて、ウェスタンブロッティング法によりプログルカゴンタンパク質を検出した。上記［細胞の培養上清の調製］の項目で調製した３種類の培養上清を、ＳＤＳ－ＰＡＧＥ（ポリアクリルアミドゲル電気泳動）法によりポリアクリルアミドゲル上でタンパク質を分子量で分離した。このタンパク質をポリアクリルアミドゲルからＰＶＤＦ膜（Bio-Rad社製）に転写させ、ブロッキング液（５％スキムミルク／ＴＢＳＴ）でブロッキングした後、一次抗体として上記６種類の抗プログルカゴンポリクローナル抗体を反応させた。一次抗体は上記の抗血清をＴＢＳＴで１０００倍に希釈して用いた。ＴＢＳＴ溶液で洗浄後、１万倍に希釈したＨＲＰ（horseradish peroxidase）標識抗ウサギ抗体を二次抗体として用い、検出はECL Prime Western Blotting Detection Reagent (GE Healthcare社製)により蛍光検出した（図７参照）。なお、コントロールとして市販の抗プログルカゴンポリクローナル抗体３種［Anti-Glicentin (Y324), Anti-GLP-1 (Y320), Anti-GLP-2 (Y322), 全て株式会社矢内原研究所製］を用いた（図８参照）。 [Western blotting using polyclonal antibody]
Proglucagon protein was detected by Western blotting using the above 6 types of anti-proglucagon polyclonal antibodies. The three types of culture supernatant prepared in the above item [Preparation of cell culture supernatant] were subjected to SDS-PAGE (polyacrylamide gel electrophoresis) method to separate proteins by molecular weight on polyacrylamide gel. This protein was transferred from a polyacrylamide gel to a PVDF membrane (manufactured by Bio-Rad), blocked with a blocking solution (5% skim milk / TBST), and then reacted with the above 6 types of anti-proglucagon polyclonal antibodies as primary antibodies. . The primary antibody used was the above antiserum diluted 1000 times with TBST. After washing with a TBST solution, a 10,000-fold diluted HRP (horseradish peroxidase) -labeled anti-rabbit antibody was used as a secondary antibody, and detection was carried out with ECL Prime Western Blotting Detection Reagent (manufactured by GE Healthcare) (see FIG. 7). ). As a control, three types of commercially available anti-proglucagon polyclonal antibodies [Anti-Glicentin (Y324), Anti-GLP-1 (Y320), Anti-GLP-2 (Y322), all manufactured by Yanaihara Institute, Ltd.] were used. (See FIG. 8).

　その結果、上記６種類の抗プログルカゴンポリクローナル抗体を用いた場合、プログルカゴンの分子量に相当する位置に目的のバンドが検出された。また、上記６種類の抗プログルカゴンポリクローナル抗体は、全て市販の抗体よりも検出感度が高いことが示された。 As a result, when the above 6 types of anti-proglucagon polyclonal antibodies were used, a target band was detected at a position corresponding to the molecular weight of proglucagon. Moreover, it was shown that all the six types of anti-proglucagon polyclonal antibodies have higher detection sensitivity than commercially available antibodies.

［モノクローナル抗体の作製］
　上記［抗原の作製］の項目で作製した３種類のプログルカゴンペプチド（１ｍｇ）とキーホールリンペットヘモシアニン（ＫＬＨ、５ｍｇ）とをそれぞれ結合したコンジュゲートをＣ５７Ｂｌ６マウスに週１回、全６回注射により免疫した後、最終の免疫から１週間後に脾臓を採取し、定法にしたがってハイブリドーマを樹立した。樹立したハイブリドーマが上記３種類のプログルカゴンペプチドに対する抗体を産生しているか、スクリーニングをＥＬＩＳＡにより行った。プログルカゴンの６１～９０番目のアミノ酸残基からなるペプチドを免疫したマウス由来のハイブリドーマを調べたところ、プログルカゴンに対する抗体を産生するハイブリドーマ１３クローンが得られ、そのうち３クローン（８１１Ａｂ、８１２Ａｂ、及び８１３Ａｂ）が確立された（図９参照）。また、プログルカゴンの１１１～１４４番目のアミノ酸残基からなるペプチドを免疫したマウス由来のハイブリドーマを調べたところ、ハイブリドーマ６クローンが得られ、そのうち４クローン（９１１Ａｂ、９１２Ａｂ、９１３Ａｂ、及び９１４Ａｂ）が確立された（図９参照）。なお、プログルカゴンの２～３０番目のアミノ酸残基からなるペプチドを免疫したマウス由来のハイブリドーマからは陽性クローンは得られなかった。 [Production of monoclonal antibodies]
Conjugates conjugated with the three proglucagon peptides (1 mg) and keyhole limpet hemocyanin (KLH, 5 mg) prepared in the above [Preparation of antigen] were injected into C57B16 mice once a week for a total of 6 times. After one immunization, the spleen was collected one week after the final immunization, and a hybridoma was established according to a conventional method. Screening was performed by ELISA to determine whether the established hybridomas produced antibodies against the above three proglucagon peptides. When hybridomas derived from mice immunized with peptides consisting of amino acid residues 61 to 90 of proglucagon were examined, 13 hybridomas producing antibodies against proglucagon were obtained, of which 3 clones (811 Ab, 812 Ab, and 813 Ab) were obtained. ) Was established (see FIG. 9). Further, when a hybridoma derived from a mouse immunized with a peptide consisting of the 111th to 144th amino acid residues of proglucagon was examined, 6 hybridoma clones were obtained, of which 4 clones (911 Ab, 912 Ab, 913 Ab, and 914 Ab) were established. (See FIG. 9). A positive clone was not obtained from a hybridoma derived from a mouse immunized with a peptide consisting of amino acid residues 2 to 30 of proglucagon.

［モノクローナル抗体の免疫反応性とエピトープの検定］
　抗体のエピトープについてポリクローナル抗体と同様に解析したところ（図９～１６参照）、ハイブリドーマ３クローン（８１１Ａｂ、８１２Ａｂ、及び８１３Ａｂ）が産生する抗体（ＳＣＣ－ＭＧＤ－８１１Ａｂ、ＳＣＣ－ＭＧＤ－８１２Ａｂ及びＳＣＣ－ＭＧＤ－８１３Ａｂ）のエピトープは、プログルカゴンの７２～７８番目のアミノ酸残基の範囲内に含まれることや（図１０～１２参照）、ハイブリドーマ３クローン（９１１Ａｂ、９１２Ａｂ、及び９１４Ａｂ）が産生する抗体（ＳＣＣ－ＭＧＤ－９１１Ａｂ、ＳＣＣ－ＭＧＤ－９１２Ａｂ及びＳＣＣ－ＭＧＤ－９１４Ａｂ）のエピトープは、プログルカゴンの１２６～１３５番目のアミノ酸残基の範囲内に含まれることや（図１３～１４及び図１６参照）、ハイブリドーマ１クローン（９１３Ａｂ）が産生する抗体（ＳＣＣ－ＭＧＤ－９１３Ａｂ）のエピトープは、プログルカゴンの１１１～１１５番目のアミノ酸残基の範囲内に含まれることが明らかとなった（図１５参照）。
　以上の結果は、プログルカゴンを特異的に認識するモノクローナル抗体７種が作製されたことを示している。 [Immunoreactivity of monoclonal antibody and epitope assay]
When the epitope of the antibody was analyzed in the same manner as the polyclonal antibody (see FIGS. 9 to 16), the antibodies (SCC-MGD-811Ab, SCC-MGD-812Ab and SCC-) produced by the hybridoma 3 clones (811Ab, 812Ab, and 813Ab) were analyzed. The epitope of MGD-813Ab) is contained within the range of amino acid residues 72 to 78 of proglucagon (see FIGS. 10 to 12), and the antibody produced by the hybridoma 3 clones (911 Ab, 912 Ab, and 914 Ab) Epitopes of (SCC-MGD-911Ab, SCC-MGD-912Ab and SCC-MGD-914Ab) are included within the range of amino acid residues 126 to 135 of proglucagon (FIGS. 13 to 14 and FIG. 16). See) Hybridoma 1 black Epitope antibodies (SCC-MGD-913Ab) that emissions (913Ab) is produced was found to be included within the scope of 111 to 115 amino acid residue proglucagon (see FIG. 15).
The above results indicate that 7 types of monoclonal antibodies specifically recognizing proglucagon were produced.

［モノクローナル抗体を用いたウェスタンブロッティング法］
　上記７種類の抗プログルカゴンモノクローナル抗体を産生するハイブリドーマの培養上清を用いて、ウェスタンブロッティング法によりプログルカゴンタンパク質を検出した。上記［細胞の培養上清の調製］の項目で調製した３種類の培養上清を、ＳＤＳ－ＰＡＧＥ（ポリアクリルアミドゲル電気泳動）法によりポリアクリルアミドゲル上でタンパク質を分子量で分離した。このタンパク質をポリアクリルアミドゲルからＰＶＤＦ膜（Bio-Rad社製）に転写させ、ブロッキング液（５％スキムミルク／ＴＢＳＴ）でブロッキングした後、一次抗体として上記７種類の抗プログルカゴンモノクローナル抗体を反応させた。一次抗体は上記の抗血清をＴＢＳＴで１０００倍に希釈して用いた。ＴＢＳＴ溶液で洗浄後、１万倍に希釈したＨＲＰ（horseradish peroxidase）標識抗マウス抗体を二次抗体として用い、検出はECL Prime Western Blotting Detection Reagent（GE Healthcare社製）により蛍光検出した（図１７参照）。 [Western blotting using monoclonal antibody]
Proglucagon protein was detected by Western blotting using the culture supernatant of the hybridoma producing the above seven types of anti-proglucagon monoclonal antibodies. The three types of culture supernatant prepared in the above item [Preparation of cell culture supernatant] were subjected to SDS-PAGE (polyacrylamide gel electrophoresis) method to separate proteins by molecular weight on polyacrylamide gel. This protein was transferred from a polyacrylamide gel to a PVDF membrane (manufactured by Bio-Rad), blocked with a blocking solution (5% skim milk / TBST), and then reacted with the above seven types of anti-proglucagon monoclonal antibodies as primary antibodies. . The primary antibody used was the above antiserum diluted 1000 times with TBST. After washing with TBST solution, an HRP (horseradish peroxidase) -labeled anti-mouse antibody diluted 10,000 times was used as a secondary antibody, and detection was performed by fluorescence detection using ECL Prime Western Blotting Detection Reagent (manufactured by GE Healthcare) (see FIG. 17). ).

　その結果、ハイブリドーマ６クローン（８１１Ａｂ、８１２Ａｂ、及び８１３Ａｂ、並びに９１２Ａｂ、９１３Ａｂ、及び９１４Ａｂ）の培養上清を用いた場合、プログルカゴンの分子量に相当する位置に目的のバンドが検出された。また、上記ハイブリドーマ６クローンのうち、４クローン（８１２Ａｂ及び８１３Ａｂ、並び９１３Ａｂ及び９１４Ａｂ）の培養上清を用いた場合、市販の抗体を用いた場合よりも高い感度でプログルカゴンを検出できることが示された。 As a result, when the culture supernatant of 6 hybridoma clones (811 Ab, 812 Ab, and 813 Ab and 912 Ab, 913 Ab, and 914 Ab) was used, a target band was detected at a position corresponding to the molecular weight of proglucagon. In addition, it was shown that proglucagon can be detected with higher sensitivity when using the culture supernatant of 4 clones (812 Ab and 813 Ab, and 913 Ab and 914 Ab) among the 6 hybridoma clones described above. It was.

　さらに、プログルカゴン１１１－１４４抗原を用いて作製したモノクローナル抗体ＳＣＣ－ＭＧＤ－９１３のエピトープは１１１－１１５領域であり、グリセンチン、グルカゴン、ＧＬＰ－１及びＧＬＰ－２配列を含まないことから、ＳＣＣ－ＭＧＤ－９１３と他の１種あるいは２種類の抗体との組み合わせによるサンドイッチ法により、プログルカゴンのみを特異的に測定することができることが明らかとなった。 Furthermore, since the epitope of the monoclonal antibody SCC-MGD-913 produced using the proglucagon 111-144 antigen is the 111-115 region and does not contain the glicentin, glucagon, GLP-1 and GLP-2 sequences, the SCC- It was revealed that only proglucagon can be specifically measured by a sandwich method using a combination of MGD-913 and one or two other antibodies.

［モノクローナル抗体遺伝子の同定］
　市販の抗体よりも検出感度が高かった上記ハイブリドーマ４クローンのうち、特に検出感度が高いハイブリドーマクローン（９１３Ａｂ）から、RNeasy Mini kit (Qiagen社製、Cat.No.74104)を用いてトータルＲＮＡを抽出した後、GeneRacer Kit （Invitrogen社製 Cat. No.L1502-01［SuperScript III RTとTOTP TA Cloning Kit for Sequencingを含む］）を用いた５’－ＲＡＣＥ法により重鎖及び軽鎖可変領域を増幅した（図１８参照）。なお、重鎖及び軽鎖可変領域の増幅に用いたプライマーを以下に示す。
mIgG-CH1プライマー；5’- ctcaattttcttgtccaccttggtgc -3’（配列番号１７で示される塩基配列）
mIgG-CLkプライマー；5’- ctcattcctgttgaagctcttgacaat -3’（配列番号１８で示される塩基配列）
5’RACEプライマー；5’- cgactggagcacgaggacactga -3’（配列番号１９で示される塩基配列）
　抗体の重鎖及び軽鎖遺伝子は、Blend Taq（TOYOBO社製、Cat. No.BTQ-101）を用いたＰＣＲにより単離し、ｐＣＲ４－ＴＯＰＯ（Invitrogen社製）ベクターを用いてクローニングした。クローニングした各ＤＮＡ断片の塩基配列は、BigDye Terminator Cycle Sequencing Kit（Applied Biosystems社製）を用い、ＤＮＡシークエンサーにて決定した（図１９及び２０参照）。また、同定した重鎖及び軽鎖可変領域遺伝子配列を基に、ＩＧＢＬＡＳＴにより検索し、重鎖及び軽鎖相補性決定領域１～３（ＣＤＲ１～３）を同定した（図１９及び２０参照）。 [Identification of monoclonal antibody genes]
Extraction of total RNA using the RNeasy Mini kit (Qiagen, Cat. No. 74104) from the hybridoma clone (913 Ab) having a particularly high detection sensitivity among the 4 hybridoma clones having a detection sensitivity higher than that of a commercially available antibody. After that, the heavy and light chain variable regions were amplified by 5′-RACE method using GeneRacer Kit (Cat. No. L1502-01 manufactured by Invitrogen [including SuperScript III RT and TOTP TA Cloning Kit for Sequencing)]. (See FIG. 18). The primers used for amplification of the heavy chain and light chain variable regions are shown below.
mIgG-CH1 primer; 5'-ctcaattttcttgtccaccttggtgc-3 '(base sequence represented by SEQ ID NO: 17)
mIgG-CLk primer; 5'-ctcattcctgttgaagctcttgacaat-3 '(base sequence represented by SEQ ID NO: 18)
5′RACE primer; 5′-cgactggagcacgaggacactga-3 ′ (base sequence represented by SEQ ID NO: 19)
The heavy and light chain genes of the antibody were isolated by PCR using Blend Taq (TOYOBO, Cat. No. BTQ-101) and cloned using a pCR4-TOPO (Invitrogen) vector. The base sequence of each cloned DNA fragment was determined with a DNA sequencer using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) (see FIGS. 19 and 20). Further, the heavy chain and light chain complementarity determining regions 1 to 3 (CDR1 to 3) were identified by searching by IGBLAST based on the identified heavy chain and light chain variable region gene sequences (see FIGS. 19 and 20).

　得られた抗プログルカゴン抗体の遺伝子配列及びそれがコードするアミノ酸配列を以下に示す。
重鎖ＣＤＲ１塩基配列：配列番号１で示される塩基配列
重鎖ＣＤＲ１アミノ酸配列：配列番号２で示されるアミノ酸配列
重鎖ＣＤＲ２遺伝子配列：配列番号３で示される塩基配列
重鎖ＣＤＲ２アミノ酸配列：配列番号４で示されるアミノ酸配列
重鎖ＣＤＲ３遺伝子配列：配列番号５で示される塩基配列
重鎖ＣＤＲ３アミノ酸配列：配列番号６で示されるアミノ酸配列
軽鎖ＣＤＲ１遺伝子配列：配列番号７で示される塩基配列
軽鎖ＣＤＲ１アミノ酸配列：配列番号８で示されるアミノ酸配列
軽鎖ＣＤＲ２遺伝子配列：配列番号９で示される塩基配列
軽鎖ＣＤＲ２アミノ酸配列：配列番号１０で示されるアミノ酸配列
軽鎖ＣＤＲ３遺伝子配列：配列番号１１で示される塩基配列
軽鎖ＣＤＲ３アミノ酸配列：配列番号１２で示されるアミノ酸配列
重鎖可変領域遺伝子配列：配列番号１３で示される塩基配列
重鎖可変領域アミノ酸配列：配列番号１４で示されるアミノ酸配列
軽鎖可変領域遺伝子配列：配列番号１５で示される塩基配列
軽鎖可変領域アミノ酸配列：配列番号１６で示されるアミノ酸配列 The gene sequence of the obtained anti-proglucagon antibody and the amino acid sequence encoded by it are shown below.
Heavy chain CDR1 base sequence: base sequence represented by SEQ ID NO: 1 heavy chain CDR1 amino acid sequence: amino acid sequence represented by SEQ ID NO: 2 heavy chain CDR2 gene sequence: base sequence heavy chain CDR2 amino acid sequence represented by SEQ ID NO: 3 Amino acid sequence represented by 4 heavy chain CDR3 gene sequence: nucleotide sequence represented by SEQ ID NO: 5 heavy chain CDR3 amino acid sequence: amino acid sequence light chain represented by SEQ ID NO: 6 CDR1 gene sequence: nucleotide sequence light chain represented by SEQ ID NO: 7 CDR1 amino acid sequence: amino acid sequence shown in SEQ ID NO: 8 light chain CDR2 gene sequence: nucleotide sequence shown in SEQ ID NO: 9 light chain CDR2 amino acid sequence: amino acid sequence shown in SEQ ID NO: 10 light chain CDR3 gene sequence: SEQ ID NO: 11 Light chain CDR3 amino acid sequence shown: SEQ ID NO: 12 amino acid sequence weight Variable region gene sequence: nucleotide sequence shown in SEQ ID NO: 13 heavy chain variable region amino acid sequence: amino acid sequence shown in SEQ ID NO: 14 light chain variable region gene sequence: nucleotide sequence shown in SEQ ID NO: 15 light chain variable region amino acid sequence: The amino acid sequence shown in SEQ ID NO: 16

　［作製した抗体を用いた新規プログルカゴン測定系の確立］
　上記抗プログルカゴンポリクローナル抗体（ＳＣＣ－ＭＧＤ－７０２Ｓ）、及び抗プログルカゴンモノクローナル抗体２種（ＳＣＣ－ＭＧＤ－８１２Ａｂ、及びＳＣＣ－ＭＧＤ－９１３Ａｂ）の計３種類の抗体について、グルカゴン、ＧＬＰ－１、及びＧＬＰ－２との交差反応性を検討した。 [Establishment of a new proglucagon measurement system using the prepared antibody]
The anti-proglucagon polyclonal antibody (SCC-MGD-702S) and two types of anti-proglucagon monoclonal antibodies (SCC-MGD-812Ab and SCC-MGD-913Ab) in total 3 types of antibodies, glucagon, GLP-1, And cross-reactivity with GLP-2 was examined.

　ＥＬＩＳＡ法を用い、上記３種類の抗体と、市販品のグルカゴン、ＧＬＰ－１、及びＧＬＰ－２との交差反応性を検討した。市販品は、化学合成品であるヒトグルカゴン（矢内原研究所社製）、ヒトＧＬＰ－１（矢内原研究所社製）、及びヒトＧＬＰ－２（矢内原研究所社製）を用いた。
　ＳＣＣ－ＭＧＤ－７０２ＳＡｂを用いたＥＬＩＳＡ法では、標識抗原にビオチル化プログルカゴン（２－３０）１０ｎｇ／ｍＬ、標準抗原にプログルカゴン（２－３０）０－２００ｎｇ／ｍＬを用いた。ＳＣＣ－ＭＧＤ－８１２Ａｂを用いたＥＬＩＳＡ法では、標識抗原にビオチル化プログルカゴン（６１－９０）１０ｎｇ／ｍＬ、標準抗原にプログルカゴン（６１－９０）０－２００ｎｇ／ｍＬを用いた。ＳＣＣ－ＭＧＤ－９１３Ａｂを用いたＥＬＩＳＡ法では、標識抗原にビオチル化プログルカゴン（１１１－１４４）１０ｎｇ／ｍＬ、標準抗原にプログルカゴン（１１１－１４４）０－２００ｎｇ／ｍＬを用いた。上記標識抗原及び標準抗原はすべて、上記［抗原の作製］の項目で作製したペプチド、若しくは上記［抗原の作製］の項目で作製したペプチドにビオチン(Sigma-Aldorich社製)を導入してビオチン化したペプチドである。 Using the ELISA method, cross-reactivity of the above three types of antibodies with commercially available glucagon, GLP-1 and GLP-2 was examined. As commercially available products, human glucagon (manufactured by Yanaihara Laboratories), human GLP-1 (manufactured by Yanaihara Laboratories), and human GLP-2 (manufactured by Yanaihara Laboratories) were used.
In the ELISA method using SCC-MGD-702SAb, biotylated proglucagon (2-30) 10 ng / mL was used as the labeled antigen, and proglucagon (2-30) 0-200 ng / mL was used as the standard antigen. In the ELISA method using SCC-MGD-812Ab, biotylated proglucagon (61-90) 10 ng / mL was used as the labeled antigen, and proglucagon (61-90) 0-200 ng / mL was used as the standard antigen. In the ELISA method using SCC-MGD-913 Ab, biotylated proglucagon (111-144) 10 ng / mL was used as the labeled antigen, and proglucagon (111-144) 0-200 ng / mL was used as the standard antigen. The labeled antigen and the standard antigen are all biotinylated by introducing biotin (manufactured by Sigma-Aldorich) into the peptide prepared in the above [Preparation of antigen] or the peptide prepared in the above [Preparation of antigen]. Peptide.

　９６穴プレートの各ウェルに、抗ウサギＩｇＧ（５μｇ／ｍＬ）(MP Biomedical社製)とマウスＩｇＧ（５μｇ／ｍＬ）(MP Biomedical社製)を各１００μＬ添加し、４℃で２４時間静置した。その後、Tween-20(Biorad社製)を含むＰＢＳで３回洗浄した。更にブロックエース(DSファーマバイオメディカル社製)３００μＬを添加し、４℃で一晩放置しブロッキングした。
　ブロッキングした９６穴プレートの各ウェルに、標識抗原溶液５０μＬを加えた後、標準抗原溶液あるいは測定試料（ＨＰＬＣ画分）５０μＬを添加し、更に検出抗体５０μＬを加え、室温で３時間放置した。その後、Tween-20を含むＰＢＳで４回洗浄後、各ウェルにストレプトアビジン－ＨＲＰ(メルク社製)を加え、室温で２時間反応させた。その後、ＰＢＳで４回洗浄し、各ウェルに発光基質溶液（Ｒ＆Ｄ社製）５０μＬを加え、室温・暗所で２０分間反応させた後、発光強度を４５０ｎｍにおける紫外部吸収で測定した。（図２１参照）。 100 μL each of anti-rabbit IgG (5 μg / mL) (MP Biomedical) and mouse IgG (5 μg / mL) (MP Biomedical) was added to each well of the 96-well plate and allowed to stand at 4 ° C. for 24 hours. . Thereafter, the plate was washed 3 times with PBS containing Tween-20 (Biorad). Further, 300 μL of Block Ace (DS Pharma Biomedical) was added and left standing at 4 ° C. overnight for blocking.
After adding 50 μL of the labeled antigen solution to each well of the blocked 96-well plate, 50 μL of the standard antigen solution or measurement sample (HPLC fraction) was added, 50 μL of the detection antibody was further added, and the mixture was allowed to stand at room temperature for 3 hours. Thereafter, after washing 4 times with PBS containing Tween-20, streptavidin-HRP (manufactured by Merck) was added to each well and reacted at room temperature for 2 hours. Thereafter, the plate was washed 4 times with PBS, 50 μL of a luminescent substrate solution (manufactured by R & D) was added to each well, and the mixture was reacted at room temperature in a dark place for 20 minutes, and then the luminescence intensity was measured by ultraviolet absorption at 450 nm. (See FIG. 21).

　その結果、ＳＣＣ－ＭＧＤ－７０２ＳＡｂ、ＳＣＣ－ＭＧＤ－８１２Ａｂ、及びＳＣＣ－ＭＧＤ－９１３Ａｂを用いたいずれのＥＬＩＳＡ系においても、グルカゴン、ＧＬＰ－１、及びＧＬＰ－２との交差反応性は認められなかった。これより、上記ＥＬＩＳＡ系はプログルカゴンに特異的であり、これらＥＬＩＳＡ系を用いることにより、プログルカゴンとグルカゴン、ＧＬＰ－１、及びＧＬＰ－２とを区別できることが示された。 As a result, no cross-reactivity with glucagon, GLP-1, and GLP-2 was observed in any ELISA system using SCC-MGD-702SAb, SCC-MGD-812Ab, and SCC-MGD-913Ab. It was. This indicates that the above ELISA system is specific to proglucagon, and proglucagon can be distinguished from glucagon, GLP-1 and GLP-2 by using these ELISA systems.

　［確立したプログルカゴン測定系と、市販のグルカゴン、ＧＬＰ－１、及びＧＬＰ－２各測定キットとの、プログルカゴン検出能の比較］
　まず、逆相ＨＰＬＣ及び質量分析を用い、ＨｕｔＵ８０細胞の条件培地内に含まれるプログルカゴンの検出を行った。
　ＨｕｔＵ８０細胞を無血清培地で培養し、得た培養上清液５００μＬをIntrada WP-RPカラム（２５０×４．６ｍｍ）(Imtakt社製)に添加した。試料の分離溶出は、溶離液Ａ（０．１％ＴＦＡ／精製水）と溶離液Ｂ（０．０８％ＴＦＡ／アセトニトリル）の混合比を３０分間で０－１００％に変化させる直線濃度勾配法で行った。流速は毎分０．７５ｍＬとし、溶出液を９６穴プレートを用いて１５秒ごとに集めた [Comparison of proglucagon detectability between established proglucagon measurement system and commercially available glucagon, GLP-1, and GLP-2 measurement kits]
First, proglucagon contained in the conditioned medium of HutU80 cells was detected using reverse phase HPLC and mass spectrometry.
HutU80 cells were cultured in a serum-free medium, and 500 μL of the obtained culture supernatant was added to an Intrada WP-RP column (250 × 4.6 mm) (Imtakt). Separate elution of the sample is a linear concentration gradient method in which the mixing ratio of eluent A (0.1% TFA / purified water) and eluent B (0.08% TFA / acetonitrile) is changed to 0-100% in 30 minutes. I went there. The flow rate was 0.75 mL per minute and the eluate was collected every 15 seconds using a 96-well plate.

　集めた各分画中のタンパク質を質量分析計(Applide Biosystems社製)で分析した結果（図２２参照）、主にフラクションＧ１－Ｇ８にプログルカゴンが検出された。フラクションＦ９－Ｆ１２、及びフラクションＧ９－Ｇ１２にもわずかであるがプログルカゴン及び、プログルカゴンのＣ端部と思われるタンパク質が検出された。 As a result of analyzing the protein in each collected fraction with a mass spectrometer (Applide Biosystems) (see FIG. 22), proglucagon was detected mainly in fractions G1-G8. Proglucagon and a protein considered to be the C-terminal part of proglucagon were detected in a small amount in fractions F9-F12 and G9-G12.

　次に、集めた各分画中のタンパク質を、上記にて作製した新規プログルカゴン測定系を用いて測定した（図２３参照）。結果、ＳＣＣ－ＭＧＤ－７０２ＳＡｂ、ＳＣＣ－ＭＧＤ－８１２Ａｂ、及びＳＣＣ－ＭＧＤ－９１３Ａｂを用いたいずれのＥＬＩＳＡ系においても、フラクションＧ１－Ｇ４に強く反応した。ＳＣＣ－ＭＧＤ－８１２Ａｂ、及びＳＣＣ－ＭＧＤ－９１３ＡｂはフラクションＧ５－Ｇ８にも強く反応したが、ＳＣＣ－ＭＧＤ－７０２ＳＡｂ、ＳＣＣ－ＭＧＤ－８１２Ａｂ、及びＳＣＣ－ＭＧＤ－９１３Ａｂのいずれも、フラクションＧ９－Ｇ１２にはほとんど反応しなかった。 Next, the proteins in each collected fraction were measured using the novel proglucagon measurement system prepared above (see FIG. 23). As a result, in any ELISA system using SCC-MGD-702SAb, SCC-MGD-812Ab, and SCC-MGD-913Ab, it reacted strongly with the fraction G1-G4. SCC-MGD-812Ab and SCC-MGD-913Ab also reacted strongly with fraction G5-G8, but all of SCC-MGD-702SAb, SCC-MGD-812Ab, and SCC-MGD-913Ab were fractions G9-G12. There was little reaction.

　更に、集めた各分画中のタンパク質を、市販の測定キットを用いて測定した。用いたキットは以下のとおりである。測定は各キットに付属の説明書に従って行った。
　１．グルカゴン測定キット（図２４参照）。
　　１－１．Glucagon EIA: YK090（矢内原研究所社製)；腸管グルカゴン、ＧＬＰ－１、及びＧＬＰ－２とは交差しないとされている。
　　１－２．Glucagon ELISA: EZGGLU-30K（Millipore社製）;グルカゴン（１－１８）、及びグルカゴン（１９－２９）とは交差しないが、オキシントモジュリンとは０．５％交差するとされている。
　２．ＧＬＰ－１測定キット（図２５参照）。
　　２－１．Total GLP-1-HS ELISA: YK161（矢内原研究所社製)；抗原はＧＬＰ－１（７－３６）であり、グルカゴン、及びＧＬＰ－２とは交差しないが、ＧＬＰ－１（１－３６）、及びＧＬＰ－１（９－３６）とは同等に交差し、ＧＬＰ－１（１－３７）、及びＧＬＰ－１（７－３７）とは約９％交差するとされている。
　　２－２．GLP-1 Total ELISA : EZGLP1T-36K（Millipore社製）;抗原はＧＬＰ－１（７－３６）であり、グルカゴン、ＧＬＰ－２、及びオキシントモジュリンとは交差しないが、ＧＬＰ－１（９－３６）とは１００％交差するとされている。
　３．ＧＬＰ－２測定キット（図２６参照）。
　　３－１．Human GLP-2 EIA : YK141（矢内原研究所社製)；抗原はＧＬＰ－２（１－３３）であり、グルカゴン、及びＧＬＰ－１とは交差しないとされている。
　　３－２．GLP-2 ELISA : EZGLP2-37K（Millipore社製）;抗原はＧＬＰ－２（１－３３）であり、ＧＬＰ－２（３－３３）とも同等に交差するとされている。 Furthermore, the protein in each collected fraction was measured using a commercially available measurement kit. The kits used are as follows. The measurement was performed according to the instructions attached to each kit.
1. Glucagon measurement kit (see FIG. 24).
1-1. Glucagon EIA: YK090 (manufactured by Yauchihara Institute); it is said that it does not intersect with intestinal glucagon, GLP-1, and GLP-2.
1-2. Glucagon ELISA: EZGGLU-30K (Millipore); does not cross glucagon (1-18) and glucagon (19-29), but 0.5% crosses oxyntomodulin.
2. GLP-1 measurement kit (see FIG. 25).
2-1. Total GLP-1-HS ELISA: YK161 (manufactured by Yanaihara Laboratories); the antigen is GLP-1 (7-36) and does not cross glucagon and GLP-2, but GLP-1 (1-36) , And GLP-1 (9-36) are crossed equally, and GLP-1 (1-37) and GLP-1 (7-37) cross about 9%.
2-2. GLP-1 Total ELISA: EZGLP1T-36K (Millipore); antigen is GLP-1 (7-36) and does not cross glucagon, GLP-2, and oxyntomodulin, but GLP-1 (9- 36) is supposed to cross 100%.
3. GLP-2 measurement kit (see FIG. 26).
3-1. Human GLP-2 EIA: YK141 (manufactured by Yanaihara Laboratories); the antigen is GLP-2 (1-33), and it is said that it does not cross glucagon and GLP-1.
3-2. GLP-2 ELISA: EZGLP2-37K (manufactured by Millipore); the antigen is GLP-2 (1-33), which is said to cross the GLP-2 (3-33) equally.

　市販のグルカゴン測定キットを用いた測定（図２４参照）では、矢内原研究所社製品及びMillipore製品共に、反応した免疫活性溶出画分は主なプログルカゴン溶出画分とは異なっていた。
　市販のＧＬＰ－１測定キットを用いた測定（図２５参照）では、矢内原研究所製品に反応した免疫活性溶出画分は主なプログルカゴン溶出画分とは異なっていた。一方、Millipore製品を用いた測定では、主なプログルカゴン溶出画分を含む広範囲な溶出画分に反応が認められた。しかし、その免疫活性は顕著に低値であり、その測定値は上記にて作製した新規プログルカゴン測定系と比べ、０．５％以下であった。
　市販のＧＬＰ－１測定キットを用いた測定（図２６参照）では、矢内原研究所製品、及びMillipore社製品共に、主なプログルカゴン溶出画分に反応が認められたが、その測定値は上記にて作製した新規プログルカゴン測定系と比べ１６％以下であった。 In the measurement using a commercially available glucagon measurement kit (see FIG. 24), the reacted immunoreactive elution fraction was different from the main proglucagon elution fraction in both the Yanaihara Institute and Millipore products.
In the measurement using a commercially available GLP-1 measurement kit (see FIG. 25), the immunologically active elution fraction reacted with the Yauchihara Institute product was different from the main proglucagon elution fraction. On the other hand, in the measurement using the Millipore product, a reaction was observed in a wide range of elution fractions including the main proglucagon elution fraction. However, the immune activity was remarkably low, and the measured value was 0.5% or less as compared with the new proglucagon measuring system prepared above.
In the measurement using a commercially available GLP-1 measurement kit (see FIG. 26), reaction was observed in main proglucagon elution fractions for both the Yanaihara Laboratory product and the Millipore product. It was 16% or less compared with the new proglucagon measurement system prepared in the above.

　これより、上記にて作製した新規プログルカゴン測定系は、未変性のプログルカゴン測定に関しては、既存のグルカゴン、ＧＬＰ－１、及びＧＬＰ－２測定キットと比較し特異性及び検出能において優れていることが示された。 Thus, the new proglucagon measurement system prepared above is superior in specificity and detectability in measuring native proglucagon compared to existing glucagon, GLP-1 and GLP-2 measurement kits. It was shown that.

　本発明の抗プログルカゴン抗体や上記他の態様の抗プログルカゴン抗体は、従来の抗プログルカゴン抗体よりも特異性が高い、又は検出感度が高いため、従来の抗プログルカゴン抗体では検出できなかった十二指腸癌等の細胞中に含まれるプログルカゴンを検出することができ、プログルカゴンを産生する十二指腸癌等の癌の有無を判定する方法や、かかる癌の再発リスクを評価する方法や、上記癌に対する抗癌剤の有効性の判定方法や、上記癌の抑制剤・治療剤のスクリーニング方法等に有利に用いることができ、プログルカゴンを産生する癌の治療薬の開発に資するものである。 The anti-proglucagon antibody of the present invention and the anti-proglucagon antibodies of the other embodiments described above have higher specificity or higher detection sensitivity than conventional anti-proglucagon antibodies, and thus could not be detected by conventional anti-proglucagon antibodies. Proglucagon contained in cells such as duodenal cancer can be detected, a method for determining the presence or absence of cancer such as duodenal cancer producing proglucagon, a method for evaluating the recurrence risk of such cancer, It can be advantageously used in methods for determining the effectiveness of anticancer agents, screening methods for the above-mentioned cancer inhibitors / therapeutics, etc., and contributes to the development of therapeutic agents for cancers that produce proglucagon.

Claims (10)

配列番号１４に示されるアミノ酸配列を有する重鎖可変領域と、配列番号１６に示されるアミノ酸配列を有する軽鎖可変領域とを備えたことを特徴とする抗プログルカゴン抗体。 An anti-proglucagon antibody comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16. 配列番号１４に示されるアミノ酸配列と８０％以上の配列同一性のアミノ酸配列を有する重鎖可変領域と、配列番号１６に示されるアミノ酸配列と８０％以上の配列同一性のアミノ酸配列を有する軽鎖可変領域とを備え、プログルカゴンに特異的に結合することを特徴とする抗プログルカゴン抗体。 A heavy chain variable region having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14, and a light chain having an amino acid sequence of 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 16 An anti-proglucagon antibody comprising a variable region and specifically binding to proglucagon. 配列番号２、４、及び６に示されるアミノ酸配列を有する重鎖相補性決定領域を含む重鎖可変領域と、配列番号８、１０、及び１２に示されるアミノ酸配列を有する軽鎖相補性決定領域を含む軽鎖可変領域とを備えたことを特徴とする抗プログルカゴン抗体。 A heavy chain variable region comprising a heavy chain complementarity determining region having the amino acid sequences shown in SEQ ID NOs: 2, 4, and 6; and a light chain complementarity determining region having the amino acid sequences shown in SEQ ID NOs: 8, 10, and 12 An anti-proglucagon antibody comprising a light chain variable region comprising 配列番号２１に示されるアミノ酸配列からなるプログルカゴンの１１１～１４４番目のアミノ酸残基内のエピトープに結合することを特徴とする抗プログルカゴン抗体。 An anti-proglucagon antibody that binds to an epitope within amino acid residues 111 to 144 of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21. 配列番号２１に示されるアミノ酸配列からなるプログルカゴンの１１１～１１５番目のアミノ酸残基内のエピトープに結合することを特徴とする請求項４記載の抗プログルカゴン抗体。 The anti-proglucagon antibody according to claim 4, which binds to an epitope within amino acid residues 111 to 115 of proglucagon consisting of the amino acid sequence shown in SEQ ID NO: 21. 請求項１～５のいずれかに記載の抗プログルカゴン抗体をコードすることを特徴とする抗プログルカゴン抗体遺伝子。 An anti-proglucagon antibody gene encoding the anti-proglucagon antibody according to any one of claims 1 to 5. 配列番号１３に示される塩基配列を有する重鎖可変領域遺伝子と、配列番号１５に示される塩基配列を有する軽鎖可変領域遺伝子とを備えたことを特徴とする請求項６に記載の抗プログルカゴン抗体遺伝子。 An anti-proglucagon according to claim 6, comprising a heavy chain variable region gene having the base sequence shown in SEQ ID NO: 13 and a light chain variable region gene having the base sequence shown in SEQ ID NO: 15. Antibody gene. 請求項１～５のいずれかに記載の抗プログルカゴン抗体を用いることを特徴とするプログルカゴンの検出方法。 A method for detecting proglucagon comprising using the anti-proglucagon antibody according to any one of claims 1 to 5. 請求項１～５のいずれかに記載の抗プログルカゴン抗体を備えたことを特徴とするプログルカゴンの検出用キット。 A proglucagon detection kit comprising the anti-proglucagon antibody according to any one of claims 1 to 5. 請求項９に記載のプログルカゴンの検出用キットを用いた、プログルカゴンを産生する腫瘍の診断方法。 A method for diagnosing a tumor producing proglucagon using the proglucagon detection kit according to claim 9.
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