WO2016002189A1 - Anticorps anti-proglucagon - Google Patents

Anticorps anti-proglucagon Download PDF

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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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proglucagon
antibody
amino acid
seq
acid sequence
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PCT/JP2015/003249
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Japanese (ja)
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徹 望月
啓一 大島
慶一 畠山
香菜子 中尾
建 山口
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静岡県
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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

La présente invention vise à procurer un anticorps anti-proglucagon qui présente une spécificité ou une sensibilité de détection élevée, un gène codant pour l'anticorps anti-proglucagon, ainsi qu'un procédé de détection et un kit de détection grâce auxquels le proglucagon exprimé dans des cellules de carcinome du duodénum, ou similaire(s) peut être détecté avec une sensibilité élevée. L'invention concerne : un anticorps anti-proglucagon comprenant une région variable de chaîne lourde ayant la séquence d'acides aminés présentée dans SEQ ID NO: 14 et une région variable de chaîne légère ayant la séquence d'acides aminés présentée dans SEQ ID NO: 16 ; un anticorps anti-proglucagon comprenant une région variable de chaîne lourde qui comporte une région déterminant la complémentarité de la chaîne lourde ayant la séquence d'acides aminés présentée dans SEQ ID NOs: 2, 4 et 6, et une région variable de chaîne légère qui comporte une région déterminant la complémentarité de la chaîne légère ayant la séquence d'acides aminés présentée dans SEQ ID NOs: 8, 10 et 12 ; un anticorps anti-proglucagon qui se lie à un épitope au sein des résidus d'acides aminés 111 à 144 du proglucagon comprenant la séquence d'acides aminés présentée dans SEQ ID NO: 12 ; un gène d'anticorps anti-proglucagon codant pour l'anticorps anti-proglucagon ; et un procédé de détection du proglucagon à l'aide de l'anticorps anti-proglucagon.
PCT/JP2015/003249 2014-06-30 2015-06-29 Anticorps anti-proglucagon WO2016002189A1 (fr)

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