WO2017047813A1 - Method for predicting prognosis and risk of developing hepatocellular carcinoma in liver cirrhosis patient - Google Patents

Method for predicting prognosis and risk of developing hepatocellular carcinoma in liver cirrhosis patient Download PDF

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WO2017047813A1
WO2017047813A1 PCT/JP2016/077683 JP2016077683W WO2017047813A1 WO 2017047813 A1 WO2017047813 A1 WO 2017047813A1 JP 2016077683 W JP2016077683 W JP 2016077683W WO 2017047813 A1 WO2017047813 A1 WO 2017047813A1
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csf1r
wfa
csr
lectin
vva
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PCT/JP2016/077683
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French (fr)
Japanese (ja)
Inventor
成松　久
晶 栂谷内
誠 雄長
裕之 梶
敦 久野
佐藤　隆
万紀 曽我部
千葉　靖典
譲 池原
田中　靖人
悦子 飯尾
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国立研究開発法人産業技術総合研究所
公立大学法人名古屋市立大学
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Priority to CN201680054422.4A priority Critical patent/CN108351359B/en
Priority to JP2017540047A priority patent/JP6779504B2/en
Publication of WO2017047813A1 publication Critical patent/WO2017047813A1/en
Priority to HK18114481.7A priority patent/HK1255344A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a method and kit for accurately grasping the progression to hepatocellular carcinoma and evaluating the prognosis and recurrence after treatment in cirrhosis, which is a serious liver disease state. More specifically, the severity of liver cirrhosis or hepatocellular carcinoma was determined using a hepatocellular carcinoma sugar chain biomarker highly correlated with the occurrence of hepatocellular carcinoma in cirrhosis (F4).
  • Liver cancer can be broadly divided into primary liver cancer that occurs in the liver and metastatic liver cancer. 90% of primary liver cancer is hepatocellular carcinoma (HCC). It is said. Patients with hepatocellular carcinoma are often infected with hepatitis C virus or hepatitis B virus as the underlying disease. After suffering from viral hepatitis, acute viral hepatitis, chronic viral hepatitis, cirrhosis As the disease progresses gradually, the liver function decreases, and as the hepatitis condition progresses and continues, the fibrosis of the liver progresses, eventually leading to cirrhosis.
  • HCC hepatocellular carcinoma
  • the carcinogenic rate also increases as the disease progresses, and the annual rate is about 0.8 to 0.9% for chronic hepatitis mild (F1) or moderate (F2).
  • chronic hepatitis is severe (F3)
  • the annual rate is 3.5%
  • cirrhosis The probability of developing cancer from F4 increases to an annual rate of 7%.
  • it may cause liver failure and death.
  • cirrhosis Even in the treatment of hepatocellular carcinoma, early detection of cancer is important because it significantly affects treatment and postoperative prognosis.
  • tests for detection of liver cancer once every three months It is necessary to receive. In order to further simplify this test, it has been required to provide a method that can accurately and easily determine the presence or absence of carcinogenesis by a blood test.
  • AFP ⁇ -fetoprotein
  • PIVKA-II protein-induced by Vitamin K absence or antagonist-II
  • the present inventors have been aiming to provide a sugar chain marker and a hepatocellular carcinoma marker that can distinguish liver disease pathologies that can detect canceration by testing body fluids such as blood, and various glycoproteins present in serum.
  • body fluids such as blood
  • various glycoproteins present in serum We have conducted research and development focusing on the changes in the sugar chain structure above.
  • the protein level of CSF1R (such as Non-patent Document 1), which has been used as a hepatocellular carcinoma marker, gradually increases from F1 to F4 as the stage progresses.
  • Non-patent Document 2 WFA lectin-binding sugar chains are hardly expressed in the F1 to F3 stages and significantly increase in patients with cirrhosis in the F4 stage.
  • Patent Document 3 The possibility of reflecting the occurrence of hepatocellular carcinoma was suggested in part of the data (Patent Document 3), but in the cirrhosis patient group, no significant difference was found depending on whether or not he had liver cancer. (Non-patent document 2). That is, the amount of WFA-binding sugar chains on CSF1R in serum was expected to be a very effective marker for discrimination of the severity of liver pathology and detection of cirrhosis.
  • JP-A-10-26622 JP-A-8-184594 International Publication 2011-007764 (WO2011 / 007764) International Publication 2014-098112 (WO2014 / 098112)
  • An object of the present invention is to provide a method for determining the risk of hepatocellular carcinoma that can accurately and easily predict the possibility of canceration by a body fluid (blood) test even for cirrhosis patients.
  • the present invention provides a method for determining the prognosis of cirrhosis for accurately and simply determining the prognosis of a patient. Specifically, among the changes in the sugar chain structure on CSF1R in body fluids such as serum of cirrhotic patients, changes to WFA-binding sugar chains that directly reflect the occurrence of hepatocellular carcinoma and / or the prognosis of cirrhosis It is intended to provide a method for accurately quantifying the proportion of
  • CSF1R-specific lectin refers to a substance that has no difference in reactivity to sugar chains on CSF1R in body fluids such as serum of healthy persons and patients.
  • CSF1R-containing sugar chain-specific lectin CSF1R-containing sugar chain-specific lectin
  • CSF1R-specific common sugar chain-binding lectin all CSF1R common sugar chain-binding lectin
  • CSF1R constitutive sugar chain structure-binding lectin CSF1R-containing sugar chain-specific lectin
  • the CSF1R-specific lectin typically corresponds to lectins such as RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA shown in Non-Patent Document 1, Fig. 3B.
  • the latter is the ratio of CSF1R containing WFA-binding sugar chains to the total CSF1R in body fluids, just like the former, and together, the “Hepatocellular carcinoma risk assessment index (WFA + -CSF1R%)” Named.
  • WFA + -CSF1R value WFA + -CSF1R level
  • the present inventors have proposed that the WFA lectin recognition sugars on CSF1R increase specifically in the development of hepatocellular carcinoma in order to increase the accuracy and stability of the measured values of the amount of WFA lectin recognition sugar chains on CSF1R and the total CSF1R amount. Elucidation of chain structure and production of highly active anti-CSF1R detection antibody were attempted. First, recombinant CSF1R was produced by cloning the CSF1R gene, and the sugar chain binding position and each sugar chain structure on CSF1R were elucidated, and the WFA lectin recognition sugar chain structure on CSF1R was identified.
  • the present inventors previously produced a recombinant WFA (hereinafter also referred to as srWFA) that has been cloned into a recombinant WFA gene and modified to prevent formation of an SS bond on the C-terminal side.
  • the srWFA was found to specifically bind to LDN sugar chains (sugar chains having “GalNAc ⁇ 1-4GlcNAc ⁇ 1-R” at the non-reducing end) (Patent Document 4).
  • the WFA lectin-recognized sugar chain structure on CSF1R may be an LDN sugar chain that srWFA specifically recognizes It was suggested that LDN sugar chains were almost demonstrated in experiments using LDN-deficient strains. Furthermore, the Glyco-Ridge method (glycopeptide / glycan structure analysis method) was applied to the recombinant CSF1R to determine the binding position of the sugar chain. It was elucidated that it binds to at least two positions at position 153 of two domains (88-209aa).
  • WFA + -CSF1R amount can also be referred to as “WFA and / or VVA (hereinafter sometimes referred to as WFA / VVA) -binding sugar chain-containing CSF1R amount”.
  • WFA / VVA VVA
  • anti-CSF1R monoclonal antibodies were prepared according to a conventional method using CSF1R as an immunogen.
  • 33 clones with high affinity for CSF1R were selected, and sandwich assay system with natural WFA lectin or monomeric recombinant WFA (srWFA) lectin together with CSF1R binding activity by direct ELISA.
  • srWFA monomeric recombinant WFA
  • hybridomas CSR-1 to CSR-33 These 33 clone antibody-producing hybridomas are referred to as hybridomas CSR-1 to CSR-33, respectively, and the monoclonal antibodies produced by each hybridoma are referred to as CSR-1 to CSR-33 antibodies, respectively. It was also found that many of the antibody recognition domains with high LDN sugar chain detection performance on CSF1R are concentrated in the second domain or the third domain.
  • the antibodies shown as detectable in the WFA-CSF1R antibody sandwich ELISA system in (Table 5), regardless of the position of the recognition domain, Are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13,
  • the CSR-26, CSR-27, and CSR-29 antibodies were superior, especially the ability to detect CSR-3, CSR-4, CSR-18, CSR-21, and CSR-30 antibodies.
  • CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5 and CSR-6 antibodies are healthy even in body fluid (serum) samples of patients with hepatocellular carcinoma against cirrhosis. It has been confirmed that an increase in CSF1R signal can be detected compared to humans.
  • a sandwich ELISA assay system combined with CSR-3 or CSR-4, CSR-18, CSR-21, CSR-30 antibody together with WFA / VVA-binding sugar chain-binding lectin, accurate WFA + -The value of CSF1R could be measured.
  • the hepatocellular carcinoma carcinogenic risk index value (C%) may be expressed as (WFA + -CSF1R%).
  • the step of measuring the total CSF1R amount in (1) is measured by a sandwich assay system using at least two types of anti-CSF1R antibodies, or purified from a test sample using anti-CSF1R antibodies and purified. The method according to [1] above, wherein the amount of CSF1R is measured.
  • the step (1) of measuring the total CSF1R amount (A) in the test sample is a method for measuring the CSF1R-specific lectin-binding sugar chain-containing CSF1R amount in the test sample.
  • CSF1R is purified from a test sample using an anti-CSF1R antibody, and the amount of purified CSF1R binding to a CSF1R-specific lectin is measured.
  • the CSF1R-specific lectin is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA. the method of.
  • the step of measuring the amount (B) of WFA / VVA-binding sugar chain-containing CSF1R in (2) is measured by a sandwich assay system containing at least a WFA / VVA-binding sugar chain-specific lectin and an anti-CSF1R antibody. Or purifying CSF1R from a test sample using an anti-CSF1R antibody, and measuring the amount of purified CSF1R that binds to WFA / VVA lectin, any one of [1] to [4] The method according to item.
  • Steps (1) and (2) are performed simultaneously using at least a CSF1R-specific lectin and a WFA / VVA lectin and an anti-CSF1R antibody, and include both the lectin and the anti-CSF1R antibody.
  • the CSF1R bound to each lectin is measured with the same assay system after the CSF1R is purified using an anti-CSF1R antibody from a test sample, or measured using the same assay system.
  • Steps (1) and (2) are performed simultaneously using at least a CSF1R-specific lectin and a WFA / VVA lectin and an anti-CSF1R antibody, and include both the lectin and the anti-CSF1R antibody.
  • the CSF1R bound to each lectin is measured with the same assay system after the CSF1R is purified using an anti-CSF1R antibody from a test sample, or measured using the same assay system.
  • a method for determining the risk of developing hepatocellular carcinoma in a cirrhotic patient comprising the steps of (1) to (3); (1) a step of calculating a risk of developing hepatocellular carcinoma (C%) of a subject who has cirrhosis according to the method according to any one of [1] to [7], (2) A WFA / VVA-binding glycan occupying in each CSF1R is calculated for each body fluid sample collected from a liver parameter cirrhosis patient who does not have hepatocellular carcinoma in advance by the same calculation step as in step (1).
  • the optimal cut-off value (M%) for the development of hepatocellular carcinoma is calculated by comparing the ratio (Cn) of the amount of CSF1R containing HC1 and the hepatocellular carcinoma incidence data obtained by following up each patient Process, (3) If the risk of developing hepatocellular carcinoma (C%) calculated in step (1) is higher than the optimal cutoff value (M%) calculated in (2), A method for determining that the risk of developing hepatocellular carcinoma is significantly high, and determining that the risk of developing is significantly low if the risk is less than the optimum cutoff value.
  • the optimal cut-off value is a value calculated based on the carcinogenic rate data obtained by following up a liver parameter cirrhosis patient who does not have a sufficient population of hepatocellular carcinoma in advance. It can be obtained by applying the minimum P-value method determined by the log rank test and excluding the upper and lower 10%.
  • the sufficient number of parameters is 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 1000, preferably 30 to 3000, 30 to 2000, 30 to 1000. More preferably, it refers to 40 to 2000, 40 to 1000, 50 to 2000, 50 to 1000, more preferably 50 to 500, and 100 to 500.
  • this determination method is a method for measuring the risk of developing hepatocellular carcinoma (C%) for predicting the risk of developing hepatocellular carcinoma in cirrhosis patients, or for diagnosing the risk of developing hepatocellular carcinoma. It can also be expressed as a method of providing material (information). [9] The method according to [8], wherein the optimum cut-off value is 35.0 ⁇ 10.0%.
  • a method for calculating a prognostic index value in a patient with cirrhosis comprising the steps of (1) and (2); (1) a step of measuring the amount of CSF1R (B) containing a WFA / VVA-binding sugar chain in a constant volume of body fluid sample (test sample) collected from a subject who has cirrhosis; (2) A step of determining the value of Bng / ml obtained in step (1) as the prognosis determination index value of the subject.
  • the prognosis determination index value (Bng / ml) may be expressed as (WFA + -CSF1R ng / ml).
  • the step of (1) measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain (B) is measured by a sandwich assay system containing at least a WFA / VVA lectin and an anti-CSF1R antibody, or a test
  • the WFA / VVA lectin is at least one lectin selected from any one of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
  • a method for determining the prognosis in a cirrhotic patient comprising the steps of (1) to (3); (1) calculating a prognostic index value (Bng / ml) of a subject who is a cirrhotic patient according to the method according to any one of [10] to [12], (2) CSF1R containing each WFA / VVA-binding glycan is obtained by the same calculation process as in step (1) on each body fluid sample collected from a liver cirrhosis patient who does not have hepatocellular carcinoma in advance.
  • the optimal cutoff value (Nng / ml) of hepatocellular carcinoma patient prognosis by comparing the amount (Bn) and cumulative survival data obtained by following up each patient; (3)
  • the prognosis determination index value (Bng / ml) calculated in step (1) is higher than the optimal cutoff value (Nng / ml) calculated in (2), A method of determining that the prognosis is significantly poor and that the prognosis of the subject is significantly good if it is less than the optimum cutoff value.
  • the optimal cut-off value is a value calculated based on a cumulative survival rate obtained by following up a liver parameter cirrhosis patient who does not have a sufficient number of hepatocellular carcinomas in advance.
  • the rate data can be obtained by applying the minimum P-value method determined by the log rank test and excluding the upper and lower 10%.
  • the sufficient number of parameters is 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 1000, preferably 30 to 3000, 30 to 2000, 30 to 1000. More preferably, it refers to 40 to 2000, 40 to 1000, 50 to 2000, 50 to 1000, more preferably 50 to 500, and 100 to 500.
  • the prognosis determination index value (Bng / ml, WFA + -CSF1R ng / ml) can be calculated as COI and used for the determination.
  • This determination method can also be expressed as a method for measuring a prognosis determination index (Bng / ml) for predicting the prognosis of a cirrhosis patient or a method for providing data for diagnosing the prognosis of a cirrhosis patient.
  • a prognosis determination index Bng / ml
  • a method for providing data for diagnosing the prognosis of a cirrhosis patient [14] The method according to [13], wherein the optimum cutoff value is 310 ⁇ 100 ng / ml.
  • An assay comprising the steps of (1) to (3) using at least one anti-CSF1R antibody selected from the group consisting of 26, CSR-27, and CSR-29 antibodies; (1) contacting the test sample with either the lectin or the anti-CSF1R antibody in a liquid phase to form a complex with CSF1R in the test sample; (2) The CSF1R complex with the lectin or antibody obtained in (1) is separated, or the other is bound to the CSF1R complex in the detection liquid phase in which the other is dissolved or dispersed.
  • a typical sandwich assay is a solid phase-liquid phase sandwich assay, particularly a sandwich ELISA, but is not limited to a sandwich ELISA, and of course is a liquid phase-liquid phase rather than a solid phase-liquid phase. May be.
  • a solid-liquid phase sandwich assay either a lectin or an antibody is provided on the capture side, and the other is dissolved or dispersed on the detection liquid phase side.
  • the “capture side” is the “solid phase surface”.
  • a lectin-antibody sandwich assay kit for detecting or quantifying CSF1R containing a WFA / VVA-binding sugar chain comprising (1) and (2); (1) WFA / VVA lectin, (2) CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13 At least one anti-CSF1R antibody selected from the group consisting of antibodies, CSR-26, CSR-27, and CSR-29.
  • the lectin-antibody sandwich assay is an assay for detecting or quantifying WFA / VVA-binding sugar chain-containing CSF1R in a body fluid sample applied to a body fluid sample derived from a subject.
  • the kit in any one of.
  • CSR-3 International accession number: NITE BP-02117
  • CSR-4 International accession number: NITE BP-02118
  • CSR-18 International accession number: NITE BP-02119
  • CSR-21 International accession number
  • CSR-30 International Accession Number: NITE BP-02121
  • an anti-CSF1R antibody produced from any one of the hybridomas or an antibody-binding fragment thereof.
  • kits for calculating a risk of developing hepatocellular carcinoma and / or a prognostic value of a subject who is a cirrhotic patient comprising the lectin of (1) and (2); (1) WFA / VVA lectin, (2) CSF1R specific lectin.
  • the kit may further contain a standard substance comprising CSF1R containing WFA / VVA-binding sugar chain and / or CSF1R containing no WFA / VVA-binding sugar chain.
  • the kit according to [22] further comprising (3); (3) An anti-CSF1R antibody or an antibody-binding fragment thereof.
  • the WFA / VVA lectin of (1) is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA
  • the CSF1R-specific lectin of (2) is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA [22] or [23] The kit according to [23].
  • Either one of the lectins of (1) or (2) is bound to a solid phase provided for capturing WFA / VVA-binding sugar chain-containing CSF1R, and the other is in a liquid phase for detection.
  • the kit may further contain a standard substance comprising CSF1R containing WFA / VVA-binding sugar chain and / or CSF1R containing no WFA / VVA-binding sugar chain.
  • the WFA / VVA lectin of (1) is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA
  • the CSF1R-specific lectin is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA
  • Anti-CSF1R antibodies are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR
  • a method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a cirrhotic patient comprising the steps of (1) and (2); (1) a step of separating and purifying CSF1R protein from a body fluid sample derived from a subject using an anti-CSF1R antibody, (2) A step of measuring the WFA / VVA-binding sugar chain content and the CSF1R-specific sugar chain content on the CSF1R separated and purified in (1).
  • a method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a cirrhotic patient comprising the steps of (1) and (2); (1) A glycoprotein containing a WFA / VVA-binding sugar chain using at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA from a body fluid sample derived from a subject.
  • anti-CSF1R antibodies are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR- 9, at least one anti-CSF1R antibody selected from the group consisting of CSR-13, CSR-26, CSR-27, and CSR-29 antibodies.
  • a method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a patient with cirrhosis uses (1) and (2) for a body fluid sample derived from the subject Performing a sandwich assay; (1) WFA / VVA lectin, (2) CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13 At least one anti-CSF1R antibody selected from the group consisting of antibodies, CSR-26, CSR-27, and CSR-29.
  • a method for determining the risk of developing hepatocellular carcinoma in a cirrhosis patient who does not have hepatocellular carcinoma comprising the steps of (1) and (2); (1) a step of measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain and the total amount of CSF1R in a body fluid sample collected from a subject who is a liver cirrhosis patient who does not have hepatocellular carcinoma; (2) calculating the ratio of the amount of CSF1R having a WFA / VVA-binding sugar chain to the total CSF1R based on the measurement value obtained in (1), (3) A WFA / VVA-binding glycan occupying in the total CSF1R is obtained from a bodily fluid sample collected from a cirrhosis patient who does not have hepatocellular carcinoma in advance by the same process as (1) and (2) above.
  • the “hepatocellular carcinoma risk determination index (WFA + -CSF1R%)” that is highly correlated with the risk of developing hepatocellular carcinoma in cirrhosis patients can be provided, and carcinogenesis according to the determination index The risk ratio could be shown. That is, a risk determination method for the occurrence of hepatocellular carcinoma in cirrhosis patients using blood samples could be provided. By using this method, even for patients with severe liver disease such as cirrhosis patients, the risk of cancer can be grasped almost accurately with a simple blood test. It became possible to reduce.
  • the present invention has been found for the first time that the amount of CSF1R containing WFA-binding sugar chains (WFA + -CSF1R value) is a "prognosis index for cirrhosis" that is highly correlated with the prognosis of cirrhosis.
  • the survival rate according to the numerical range could be shown. That is, it was possible to provide an accurate method for determining the prognosis of a cirrhotic patient using a blood sample.
  • VVA lectin reagent is effective together with a monomeric recombinant WFA lectin reagent as a lectin for detecting a WFA-binding sugar chain on CSF1R of the present invention.
  • a plurality of anti-CSF1R monoclonal antibodies having high binding activity could be provided as antibodies for detecting CSF1R containing WFA and / or VVA (WFA / VVA) -binding sugar chains.
  • Immunohistochemical staining analysis CSF1R and WFA epitope expression in hepatocellular carcinoma tissue
  • Immunohistochemical staining analysis Histochemical staining analysis using HCC tissue array (anti-CSF1R antibody and WFA) Analysis process flowchart Shows the breakdown of 214 HCV-derived hepatitis patients who participated in this cohort. In this example, cirrhosis patients who did not develop hepatocellular carcinoma (non-HCC-LC patients, 56 patients) were selected, and 45 other non-HCC-LC patients randomly selected as a validation cohort Groups were evaluated together. The optimal cutoff value of WFA + -CSF1R% for predicting the cumulative cancer rate by the minimum P-value method determined by the log rank test.
  • LDN (+) rCSF1R is rCSF1R expressed in HEK293 cells, and LDN ( ⁇ ) rCSF1R is expressed in knockout cells.
  • Glycopeptide and glycosylation position confirmed by Glyco-Ridge method in CSF1R glycoprotein (derived from mouse myeloma NS0 cells). Identified by IGOT (131120CSF-RTL-Am + GOT-dd10-35g-01).
  • Glycopeptide and glycosylation position confirmed by Glyco-Ridge method in CSF1R glycoprotein (standard CSF1R glycoprotein).
  • the core peptide candidate may include an unintended degradation sequence in the predicted trypsin degradation sequence.
  • anti-CSF1R antibodies CSR-3, CSR-4, CSR-5, CSR-6, CSR-18 antibodies
  • Antibody-VVA lectin sandwich ELISA system using antibody CSR-18. Similar to WFA, VVA can be distinguished from serum pool serum (K1, K2, K3) of patients with hepatocellular carcinoma and pool serum of healthy individuals by ELISA together with anti-CSF1R antibody.
  • Total CSF1R measurement was performed by sandwich ELISA with anti-CSF1R antibody-anti-CSF1R antibody using rCSF1R (LDN +) and rCSF1R (LDN-) prepared to the same concentration (dilution series).
  • Detection was performed in a buffer system (BSA dilution: A) or a serum system (10% NHS dilution: B), respectively.
  • BSA dilution: A a buffer system
  • a serum system (10% NHS dilution: B) a serum system
  • Total CSF1R was measured by antibody-antibody sandwich ELISA (Total).
  • Total CSF1R was performed by sandwich ELISA using antibody-each common sugar chain probe lectin (each lectin name notation).
  • rCSF1R LDN +
  • rCSF1R LDN-
  • rCSF1R LDN-
  • Detection was performed in a buffer system (BSA dilution: A, C, E) or serum system (10% NHS dilution: B, D, F), respectively. As a result, the total CSF1R amount is detected almost in the same manner.
  • the pathophysiology from chronic hepatitis to cirrhosis is based on the pathological morphology of fibrotic changes in the liver Gleason region and liver lobule, and is mild (F1), moderate (F2), severe (F3), liver cirrhosis ( F4) (New Inuyama classification method).
  • cirrhosis refers to a case where the degree of liver fibrosis is in a state corresponding to F4.
  • the pathological conditions include hepatic dysfunction due to decreased hepatocytes and the formation of esophageal gastric varices due to increased portal pressure, and severe cases include jaundice, hepatic encephalopathy, ascites retention, and gastrointestinal bleeding. And hepatocellular carcinoma appears with a probability of about 7% per year.
  • the present invention relates to a “hepatocellular carcinoma onset risk index” and a hepatocyte for predicting the risk of developing hepatocellular carcinoma using a body fluid (serum etc.) sample in a cirrhotic patient who is such a serious liver disease. Is to provide a “cirrhosis prognostic index” for predicting the prognosis (survival rate) of patients with cirrhosis who have not developed cancer.
  • the “subject” refers to a person who is subjected to a test, that is, a person who provides a sample to be described later, and is a cirrhosis patient, preferably a cirrhosis patient not suffering from hepatocellular carcinoma.
  • the subject includes a patient suffering from liver disease (acute hepatitis, chronic hepatitis, liver fibrosis, cirrhosis), a hepatocellular carcinoma patient, or a healthy person.
  • the “sample” is a body fluid collected from the subject and used for the determination method of the present embodiment, and the “body fluid” refers to a liquid biological sample collected from the subject.
  • blood including serum, plasma and interstitial fluid
  • bile lymph fluid
  • tissue fluid tissue fluid, intercellular fluid
  • body cavity fluid extract of each tissue or cell
  • pleural effusion sputum
  • cerebrospinal fluid cerebrospinal fluid
  • tear fluid Nasal discharge saliva, urine, vaginal fluid, semen and the like.
  • serum, plasma and bile are preferred.
  • the body fluid may be used after performing treatment such as dilution or concentration of a sample collected from a subject, addition of a blood coagulation inhibitor such as heparin as necessary, or such pretreatment. It may be used as it is.
  • the body fluid may be collected based on a known method in the field.
  • a known blood collection method may be followed.
  • peripheral blood it can be collected by injection into a peripheral vein or the like.
  • the bodily fluid may be used immediately after collection, or may be used after being frozen or refrigerated for a certain period of time and then subjected to processing such as thawing as necessary.
  • serum when serum is used, a sufficient amount of hepatoma can develop in patients with cirrhosis or cirrhosis if a volume of 10 ⁇ L to 100 ⁇ L, 20 ⁇ L to 80 ⁇ L, 30 ⁇ L to 70 ⁇ L, 40 ⁇ L to 60 ⁇ L, or 45 ⁇ L to 55 ⁇ L is used.
  • the value of CSF1R molecule (WFA + -CSF1R) that binds to WFA and the total amount of CSF1R necessary for quantitatively predicting prognosis can be measured.
  • Hepatocellular carcinoma risk index for cirrhosis patients is important to measure the amount of CSF1R molecule (WFA + -CSF1R) containing a sugar chain that binds to WFA and / or VVA.
  • WFA + -CSF1R CSF1R molecule
  • the value of (WFA + -CSF1R) can be called a prognostic marker for cirrhosis patients as described later, it does not predict the risk of developing hepatocellular carcinoma in cirrhosis patients.
  • the ratio of (WFA + -CSF1R) in the total CSF1R” for predicting the risk of developing hepatocellular carcinoma in cirrhosis patients is expressed in “%”. As long as it is a notation showing a ratio, it may be a decimal notation or a fraction notation.
  • a decimal or fractional representation when 100% is 1 (or a numerical representation of the CSF1R containing WFA-binding sugar chain as a relative ratio to the total CSF1R when the total CSF1R is 1) Or a value expressed in thousandths ( ⁇ ).
  • ⁇ a value expressed in thousandths
  • CSF1R-specific lectin refers to those that have no difference in reactivity to sugar chains on CSF1R in body fluid (serum) of healthy individuals and cirrhotic patients. In other words, it means a lectin that is reactive to sugar chains on all CSF1R proteins in body fluid (serum).
  • lectins such as RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA shown in Non-Patent Document 1 and FIG.
  • CSF1R containing a WFA-binding sugar chain specifically binds not only to WFA but also to a VVA lectin.
  • lectins there is a high possibility that they contain a sugar chain structure having a common or similar apoptotic structure portion. It is thought that there are several structures, but other experimental results of the present invention strongly suggest that one of them is an “LDN sugar chain”.
  • WFA and / or VVA (hereinafter referred to as“ WFA / VVA ”) binding sugar chain” is referred to as “WFA binding sugar chain-containing CSF1R amount” as “WFA / VVA-binding sugar chain-containing CSF1R amount ".
  • the calculation method of the onset risk value of hepatocellular carcinoma of cirrhosis patients of the present invention Measure the total CSF1R amount (A) and WFA / VVA-binding sugar chain-containing CSF1R amount (B) of a certain volume of body fluid sample (also referred to simply as a test sample) collected from a subject derived from a cirrhotic patient.
  • Hepatocellular carcinoma risk index of the present invention (WFA + -CSF1R%) is measured beforehand the WFA + -CSF1R% of pre sufficient population parameter of hepatocellular carcinoma unaffected cirrhosis patients, and follow-up of patients
  • the presence or absence of hepatocellular carcinoma can be accurately determined by applying the minimum P-value method determined by the log rank test, for example, and excluding the upper and lower 10% to determine the optimal cut-off value of the carcinogenic rate .
  • the minimum P-value method determined by the log rank test, for example, and excluding the upper and lower 10% to determine the optimal cut-off value of the carcinogenic rate .
  • the more accurate the population the more accurate it will be.
  • considering the time and effort required to secure the population in general, there are several tens to hundreds of cases. is there.
  • “sufficient parameters” means 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 1000, preferably 30 to 3000, 30 to 2000. 30-1000 examples, more preferably 40-2000 examples, 40-1000 examples, 50-2000 examples, 50-1000 examples, more preferably 50-500 examples, 100-500 examples.
  • the carcinogenic risk index WFA + -CSF1R% can be predicted to be extremely high at or above the optimal cutoff value.
  • the training set and validation set for the 101 population were 35.0%.
  • the 5-year cumulative incidence was 75% in the high-value group and 30-42% in the low-value group.
  • the optimal cutoff value for hepatocellular carcinoma risk index in patients with cirrhosis is 30.0 to 40.0. %, At least in the range of 25.0-45.0%. That is, when the hepatoma risk index (WFA + -CSF1R%) in cirrhosis patients is 35.0 ⁇ 10.0% or more, preferably 35.0 ⁇ 5.0% or more, it can be determined that hepatocellular carcinoma is significantly developed. On the other hand, if it is less than 35.0 ⁇ 10.0%, preferably less than 35.0 ⁇ 5.0%, it can be determined that the probability of developing hepatocellular carcinoma is significantly low.
  • the cut-off value is a value that separates a patient group (subject group A) suffering from a certain disease from a non-patient group (subject group B). These are considered to vary slightly depending on the size (number) of the population to be measured, but depending on the calculation method that is statistically standard, it is possible to set an optimal cutoff value by itself. I can do it.
  • the above-described general-purpose method is applied to the minimum P-value method determined by the log rank test, and the optimal cutoff value of the carcinogenic rate is calculated by excluding the upper and lower 10%. This method can also be used.
  • the cutoff value can be set using a ROC curve (Receiver Operator Characteristic Curve).
  • the point at which the distance from the upper left corner is minimum can be set as a cutoff value.
  • the cut-off value can also be calculated by a method using Youden index (a point where “sensitivity + specificity ⁇ 1” is the maximum value is called Youden index).
  • it can be applied to the minimum P-value method determined by the Cox regression method, and the optimal cutoff value of the survival rate can be obtained by excluding the upper and lower 10%.
  • Prognostic index for patients with cirrhosis WFA + -CSF1R ng / ml
  • the method for calculating the prognostic index value in the cirrhosis patient of the present invention “Bng / ml (WFA) obtained in the step of measuring the amount of CSF1R (B) containing a WFA / VVA-binding sugar chain in a body fluid sample (test sample) collected from a subject who is a cirrhotic patient. + -CSF1R ng / ml) ”is used as the prognostic index value of the subject.
  • the prognostic index (WFA + -CSF1R ng / ml) of the present invention was determined in advance by measuring WFA + -CSF1R ng / ml of liver cirrhosis patients not affected by hepatocellular carcinoma with a sufficient population, and the patients were kept for at least 5 years. By following up and applying the existence presence / absence data to the minimum P-value method determined by the log rank test, excluding the upper and lower 10%, the optimum cutoff value of the survival rate can be obtained accurately.
  • the index for predicting the prognosis of cirrhosis patients is expressed in units of “ng / ml”.
  • WFA-binding sugar chain-containing contained in the body fluid (serum) sample of the subject. Because it shows a typical numerical value representing CSF1R amount, it may be expressed in other units such as mg / ml, w / v%, logarithmic display, or a specific calculation formula (arithmetic formula) Other notations such as the value of the operation value converted by the coefficient may be used. Further, a cut-off index (COI) can be calculated from these cut-off values. COI is calculated as a ratio to the cutoff value, and 1.0 is the boundary between positive and negative judgments.
  • COI cut-off index
  • a cut-off index is often calculated from an actual value and used in order to eliminate the fluctuation of the value due to the measured value.
  • the optimal cutoff value of 310 ng / ml of WFA + -CSF1R was derived by the minimum P-value method determined by the log rank test, and the survival rate was examined by a time-dependent ROC curve.
  • the WFA + -CSF1R value was 310 ng.
  • the optimal cutoff value of the prognostic index in patients with cirrhosis is 260 to 360 ng / ml, at least It can be said that it is in the range of 210 to 410 ng / ml.
  • the prognostic index (WFA + -CSF1R ng / ml) in patients with cirrhosis is 310 ⁇ 100 ng / ml or more, preferably 310 ⁇ 50 ng / ml or more, it can be judged that the prognosis (survival rate) is significantly poor. . On the contrary, if it is less than 310 ⁇ 100 ng / ml, preferably less than 310 ⁇ 50 ng / ml, it can be judged that the prognosis is significantly better.
  • the optimum cutoff value may be determined using the other cutoff value calculation method described above.
  • CSF1R is a colony stimulating factor 1 (CSF1) receptor (macrophage stimulating factor-1 receptor) essential for the differentiation of monocyte cells And present on the cell surface.
  • CSF1R colony stimulating factor 1
  • macrophage stimulating factor-1 receptor macrophage stimulating factor-1 receptor
  • CSF1R is known to be cleaved by an extracellular metalloprotease with cell activation (activated), and blood CSF1R is considered to be a cleaved extracellular domain.
  • CSF1R consists of 972 amino acids (SEQ ID NOs: 1 and 2) and has been conventionally used as a hepatocellular carcinoma marker.
  • a plurality of recombinant CSF1Rs are commercially available, for example, Fc fusion type (NS0); R & D Systems).
  • a primer was designed using the known CSF1R base sequence (SEQ ID NO: 1), and human monocytic leukemia
  • the CSF1R gene was cloned using a cell line (THP-1) -derived cDNA as a template, and “standard recombinant CSF1R” was produced using HEK293 cells as a host.
  • the sugar chain structure on the standard recombinant CSF1R is as shown in (2-4) below (FIG. 13).
  • anti-CSF1R antibody In order to detect the total amount of CSF1R in the sample, it is preferable to use an anti-CSF1R monoclonal antibody (hereinafter also simply referred to as “anti-CSF1R antibody”).
  • Anti-CSF1R antibodies are commercially available anti-CSF1R antibodies, for example, anti-CSF1R mAb Cat # MAB3292 (R & D Systems) as a solid-phase antibody and biotinylated anti-CSF1R pAb Cat # BAF329 (R & D) as a detection antibody (for Total CSF1R). Systems) and the like can be used, but can be produced according to a conventional method using CSF1R as an immunogen.
  • an anti-CSF1R monoclonal antibody was also newly produced.
  • a number of anti-CSF1R monoclonal antibodies were produced according to a conventional method using CSF1R as an immunogen.
  • 33 clones having high affinity for CSF1R were selected, and together with CSF1R binding activity by direct ELISA, WFA / VVA on CSF1R in a sandwich assay system with natural WFA lectin or srWFA lectin
  • the detection performance of the binding sugar chain was verified, and a plurality of anti-CSF1R monoclonal antibodies with particularly high detection performance were further selected.
  • antibodies excellent in detection by the WFA / VVA-CSF1R antibody sandwich ELISA system are CSR-3, CSR-4, CSR-18, CSR-, regardless of the recognition domain. 21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13, CSR-26, CSR-27, CSR-29 antibodies are excellent.
  • CSR-3, CSR-4, CSR-18, CSR-21, and CSR-30 were highly capable of detecting CSF1R molecules containing WFA / VVA-binding sugar chains.
  • WFA / VVA lectin which is an LDN sugar chain-binding lectin, and is also referred to as a natural WFA lectin, a recombinant WFA lectin (SEQ ID NO: 4), or a monomeric recombinant WFA lectin (srWFA.
  • VVA lectin is preferred, and monomeric recombinant WFA lectin that binds specifically to LDN sugar chains is most preferred.
  • the WFA lectin recognition sugar chain structure on CSF1R that increases in the body fluid (serum) of cirrhosis patients is a sugar chain (LDN sugar chain) with "GalNAc ⁇ 1-4GlcNAc ⁇ 1-R" at the non-reducing end.
  • LDN sugar chain sugar chain
  • the sugar chain structure and sugar chain position on the standard rCSF1R produced in the present invention were determined, and rCSF1R produced by the LDN sugar chain-deficient strain lost the ability to bind to the WFA lectin.
  • Natural WFA is a lectin derived from leguminous Wisteria floribunda (Nodafuji), and the sugar binding specificity includes (terminal) N-acetylgalactosamine (GalNAc) sugar chain, particularly LacdiNAc (LDN: GalNAc1-3GlcNAc-R) is known to bind to the sugar chain structure.
  • GalNAc N-acetylgalactosamine
  • LDN LacdiNAc
  • Natural WFA has a structure in which the amino acid sequence in which 13 amino acids on the C-terminal side are lost from the full length of WFA consisting of SEQ ID NO: 4 is dimerized (Patent Document 4).
  • normal recombinant WFA (SEQ ID NO: 4) is obtained as a mixture of a dimer and a monomer, and is known to have the same sugar chain binding activity as natural WFA, including LDN sugar chain binding. Therefore, recombinant WFA can be used as the WFA lectin of the present invention in the same manner as natural WFA. Moreover, since WFA derivatives, such as a WFA reductant described in Patent Document 4, and other WFA variants have LDN binding activity, they can be used in the same manner.
  • the “srWFA” actually used in this example was monomerized by modifying Cys at position 272 of SEQ ID NO: 3 to Ala, and further an N-linked sugar chain that is not required for sugar chain-binding activity. Asn at position 146 was introduced into Gln and a large amount was produced in yeast.
  • VVA lectin is a lectin derived from the leguminous Vicia villosa (Hairy Vetch) seeds, and is a glycoprotein having a molecular weight of 102 kDa to 144 kDa. As sugar-binding specificity, it is known to bind to (terminal) N-acetylgalactosamine (GalNAc).
  • CSF1R-specific lectin-binding sugar chain-containing CSF1R amount that can be substituted for “total CSF1R amount” in body fluid
  • CSF1R-specific lectin It is also a “glycan structure-binding lectin”, which is reactive to all CSF1R constitutively contained in body fluid (serum) regardless of whether it is a healthy person or a patient with cirrhosis. It has a lectin.
  • the “total CSF1R amount” is measured by measuring the amount of the “CSF1R-specific lectin” reactive sugar chain on the CSF1R in the body fluid sample. It can. That is, by determining the content ratio of “WFA and / or VVA-binding sugar chain” and “CSF1R-specific lectin-binding sugar chain” on CSF1R in a body fluid sample, hepatocellular carcinoma in liver cirrhosis patients Risk (WFA + -CSF1R%) can be calculated.
  • CSF1R-specific lectins are RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA, which are also shown in Non-Patent Document 1, Fig. 3B. And other lectins.
  • RCA120 (RCA I; Ricinus communis Agglutinin I; derived from castor bean) has Gal or GalNAc specificity
  • DSA Natural stramonium; derived from datura
  • PolyLacNAc poly-N-acetyllactosamine
  • PHA-E4 Phaseolus vulgaris; derived from kidney beans
  • SNA EBL; Elderberry Balk (Sambucus nigra) Lectin; derived from elderberry) has Sialyl-Gal or Sialyl-GalNAc specificity
  • SSA Sudbucus sieboldiana; derived from Japanese elderberry
  • TJA-I Trichosanthes japonica; derived from Kikarasuuri
  • TJA-I Trichosanthes japonica; derived from Kikarasuuri
  • Measurement method and kit for the same used in the present invention (3-1) Use of measurement method and kit of the present invention Anti-CSF1R antibody newly provided in the present invention, and WFA and / or VVA lectin together with anti-CSF1R antibody
  • the method and kit for measuring CSF1R molecule, WFA + -CSF1R molecule are the risk index (WFA + -CSF1R%) and prognosis index (WFA + -CSF1R ng / ml) of hepatocellular carcinoma in cirrhosis patients in the present invention. Can be used to determine the prognosis of cirrhosis and the risk of developing hepatocellular carcinoma.
  • each kit of the present invention a WFA / VVA-binding sugar chain-containing CSF1R and / or a WFA / VVA-binding sugar chain-free CSF1R standard substance is preferably provided for positive control or negative control.
  • the components of each kit may be in the form of a solid such as a powder, in the form of a solution dissolved or dispersed in a buffer solution, or in a state of being bound to an assay substrate or beads.
  • a buffer for dissolution or dispersion can be added to the components of the kit.
  • the WFA + -CSF1R molecule that is, the WFA / VVA-binding sugar chain on CSF1R
  • the WFA + -CSF1R molecule measurement of the present invention is performed.
  • the method and kit can also be used for determining the severity of liver disease.
  • CSF1R molecules have been conventionally used as liver disease markers, liver cancer markers, etc.
  • the CSF1R molecule measurement method and kit of the present invention should be used for liver disease diagnosis or hepatocellular carcinoma diagnosis, etc. You can also.
  • the amount of CSF1R in the sample can be directly measured by ELISA using an anti-CSF1R antibody, but can be indirectly measured by measuring the amount of CSF1R-specific lectin-binding sugar chain on CSF1R.
  • the sample is substantially The "total CSF1R amount" can be measured.
  • CSF1R is separated and purified from a body fluid sample by applying a conventional protein purification method such as an anti-CSF1R antibody affinity column, and the reaction amount of WFA and / or VVA lectin and the amount of CSF1R-specific lectin reaction against the CSF1R are measured simultaneously or separately.
  • a conventional protein purification method such as an anti-CSF1R antibody affinity column
  • the reaction amount of WFA and / or VVA lectin and the amount of CSF1R-specific lectin reaction against the CSF1R are measured simultaneously or separately.
  • (3-2) Lectin-antibody sandwich immunological detection method Basically, among the protocols used for the sandwich detection method using two kinds of antibodies, it can be applied only by replacing one antibody with a lectin. Therefore, this method can be applied not only to the existing ELISA method but also to automation using an automatic immunodetection device. The only thing to consider is the reaction between the antibody used in the sandwich and the lectin. The antibody has at least two N-linked sugar chains. Therefore, when the lectin used recognizes a sugar chain on the antibody, background noise due to the binding reaction occurs during sandwich detection.
  • a method of introducing a modification into the sugar chain part on the antibody or a method of using only a Fab that does not contain a sugar chain part can be considered.
  • methods for modifying the sugar chain include Chen S et al. Nat Methods. 4, 437-44 (2007) and Consale MA et al. J Proteome Res. 8, 595-602 (2009). Include, for example, Matsumoto H et al. Clin Chem Lab Med 48, 505-512 (2010).
  • a detection system is constructed with a combination of high sensitivity and low background noise.
  • WFA / VVA lectin VVA lectin can be used in addition to commercially available WFA lectin (natural WFA), recombinant WFA, and LDN sugar chain-specific monomer srWFA.
  • anti-CSF1R antibody an anti-CSF1R monoclonal antibody is preferably used, and CSR-1 to 30, preferably CSR-3, CSR-4, CSR-18, CSR-, collected from the 30 clone hybridoma obtained in the present invention. 21 and CSR-30, or commercially available anti-CSF1R monoclonal antibodies can be used.
  • the anti-CSF1R monoclonal antibody may be an antibody fragment such as Fab or F (ab ′) 2 as long as it has an antigen recognition site. It may be a -specific antibody, an antibody obtained by artificially recombining the sequence of the antigen recognition site to produce an antibody of another species (such as a humanized antibody), or an antibody fragment thereof. Furthermore, a substance such as a phage display antibody (phage display) may be used as long as it has a binding property to an antigen.
  • phage display antibody phage display
  • a detection system using a biotin-avidin reaction using a biotin-labeled lectin is simpler and preferable than using a secondary antibody for lectin detection. Specifically, after reacting biotin-labeled WFA lectin, VVA lectin, etc., discard the solution, wash, react with HRP-labeled streptavidin solution, discard the reaction solution, wash, and then use the TMB substrate solution What is necessary is just to observe color development.
  • the detection antibody or lectin is labeled with a fluorescent substance instead of a biotin label, thereby constructing a system (system that does not depend on chemiluminescence) that directly detects the binding of the antibody or lectin.
  • CSF1R-specific lectin RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA and the like are candidates.
  • an antibody-antibody sandwich assay using two types of anti-CSF1R antibodies to measure “total CSF1R amount” RCA120 (and / or DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL) , ConA
  • anti-CSF1R antibodies can be used for lectin-antibody sandwich assays.
  • body fluid samples preferably anti-CSF1R antibody If the CSF1R antibody is overlaid, it can be said that “WFA + -CSF1R%” can be obtained in one measurement.
  • CSF1R having a sugar chain that specifically reacts with WFA / VVA lectin for example, a method of measuring CSF1R specifically binding to WFA / VVA lectin, specifically, fixing WFA / VVA lectin
  • a method for collecting and separating by a column or array, and a means for measuring CSF1R specifically, direct detection by mass spectrometry of CSF1R glycoprotein or using an antibody against the CSF1R (including fragment) .
  • a complex formed by a lectin-antibody sandwich can be formed first, and then separated by the method described later to detect this.
  • the CSF1R glycoprotein (glycopeptide) having a WFA / VVA-binding sugar chain can also be detected and quantified by Western blotting.
  • CSF1R glycoprotein is separated and purified from a body fluid (serum) sample obtained from a subject using an antibody against CSF1R (including fragments).
  • the separated and purified CSF1R glycoprotein is applied to a lectin array, a fluorescently labeled anti-CSF1R antibody is added to form a complex, and the fluorescence intensity (that is, CSF1R glycoprotein of each lectin spot) is detected by an array scanner. The amount of binding to each lectin) is measured.
  • the CSF1R marker having a sugar chain that specifically binds to the target WFA / VVA lectin the CSF1R marker that binds to the CSF1R-specific lectin, and all CSF1R markers can be measured.
  • the CSF1R glycoprotein obtained in (1) can also be detected and quantified by a lectin blot method using WFA / VVA lectin.
  • LC-MS quantitative mass spectrometry
  • immunoassay enzyme activity measurement
  • capillary electrophoresis liquid chromatography
  • HPLC liquid chromatography
  • LC-MS enzyme immunoassay using a monoclonal antibody or polyclonal antibody specific for CSF1R glycoprotein having a sugar chain that specifically reacts with WFA (VVA) lectin or a fragment thereof
  • two-antibody sandwich ELISA method gold colloid method, radioimmunoassay method, latex agglutination immunoassay method, fluorescence immunoassay method, western blotting method, immunohistochemistry method, surface plasmon resonance method (SPR method) or quartz crystal microbalance (QCM) method
  • QCM quartz crystal microbalance
  • the detection method by mass spectrometry can be carried out as follows. Marker glycopeptides and glycoproteins are glycopeptides that have LacdiNAc (LDN) sugar chains using a mass spectrometer as a detector for samples collected with the probe lectin that binds to the sugar chain or the prepared anti-CSF1R antibody. Can be detected.
  • the marker glycopeptide is preferably detected by excising the sugar chain of the collected glycopeptide, separating it by liquid chromatography (LC), and then eluting the peptide directly and directly on-line to a mass spectrometer (MS). Can be introduced and detected.
  • mass spectra can be acquired by acquiring MS / MS spectra using a fracture method such as collision-induced dissociation (CID), and only when preselected ions are detected. It is also possible to detect a plurality of fragment ions that are broken by a method such as single reaction monitoring or multi-reaction monitoring. Furthermore, by synthesizing the core peptide part of the liver cancer marker glycopeptide to the analysis sample, adding the target peptide that has incorporated a stable isotope into a part of it and causing a mass difference, and comparing the signal intensity of each Relative or absolute quantitative analysis can also be performed. In a simple manner, the signal intensity of the detected ions can be easily quantified by comparing between a plurality of samples or a standard sample.
  • CID collision-induced dissociation
  • WFA / VVA lectin and RCA120 are allowed to act on the purified CSF1R derived from the sample liquid sample, respectively.
  • the amount of CSF1R bound to WFA / VVA lectin in the sample fluid sample and CSF1R specific The amount of CSF1R bound to lectin can be measured respectively.
  • the amount ratio of WFA / VVA binding sugar chain containing CSF1R with respect to the total amount of CSF1R can be determined.
  • a capillary electrophoresis apparatus an apparatus using microfluidics technology / separation / detection technology, such as ⁇ TASWako i30 (Wako Pure Chemical Industries, Ltd.), and the like can be used. .
  • the clinical test method, measurement method, and analysis method used in this example will be described below.
  • the serum samples used in this example were stored frozen at ⁇ 80 ° C. and thawed until use for testing.
  • the clinical tests performed in this example were platelet count, prothrombin activation time (PT), serum aspartate aminotransferase concentration (AST), serum alanine aminotransferase concentration (ALT), serum albumin, serum total bilirubin ( T.bil) was conducted in a conventional manner.
  • Serum alpha-fetoprotein (AFP), AFP-LCA lectin fraction (AFP-L3,%), and vitamin K-dependent coagulation factor precursor II (PIVKA-II) were also measured in the same sample at the first visit.
  • Serum AFP was measured using HISCL-2000i (Sysmex), and PIVKA-II was measured using a Lumipulse Presto II (Fujirebio) automated chemiluminescent enzyme immunoassay device (CLEIA).
  • CLIA Lumipulse Presto II automated chemiluminescent enzyme immunoassay device
  • Conventional AFP-L3 was measured by lectin affinity chromatography and liquid phase binding assay using an automated immunoassay device ⁇ TASWako i30 (Wako Pure Chemical Industries).
  • HCC Observation period and treatment of HCC
  • Patient follow-up was performed at least every 6 months from the start using tumor markers AFP, PIVKA-II, AFP-L3, and ultrasonography, CT, and magnetic resonance imaging.
  • the first year of follow-up after HCC treatment is imaged every 3 months, during which time pneumonia, sepsis, liver-related deaths including HCC, and deaths due to liver failure including esophageal varices bleeding was analyzed.
  • HCC was treated according to Japanese guidelines. Patients were first evaluated for surgical indications, and if they refused or were ineligible for surgical treatment, local coagulation (LAT) with percutaneous ethanol injection, or more recently radiofrequency ablation (RFA), was performed. None of the patients received a liver transplant.
  • the follow-up period for each HCC patient started between 1998 and 2014 and continued until patient death or August 2014. The follow-up period ranged from 1 to 195 months (median 60 months).
  • Predictors of survival and cumulative carcinogenesis were examined for age, gender, albumin level, platelet count, Fib4, APRI, AFP, PIVKA-II, and AFP-L3.
  • the usefulness of WFA + -CSF1R and WFA + -CSF1R% was examined by time-dependent ROC analysis.
  • the correlation between continuous variables was quantified by Spearman's rank correlation coefficient. P ⁇ 0.05 was considered statistically significant.
  • Statistical analysis was performed using statistical analysis software such as SPSS.20, R 2.14.0 (survival ROC package) and Windows Excel 2010.
  • Example 1 Analysis of CSF1R glycoprotein by immunohistochemical staining method Using specimens (frozen specimens or paraffin-embedded specimens) sliced from tissues of patients with various liver diseases, particularly cirrhosis or liver cancer, Expression by immunohistochemical staining with lectin or antibody can be examined. Therefore, we examined the expression of WFA and CSF1R in liver cancer tissues using tissue array slides (made from formalin-fixed paraffin-embedded blocks). The tissue array slide was deparaffinized, washed with distilled water, and antigen-activated by heating for 5 minutes in a microwave oven (microwave oven) in 100 mM citrate buffer (pH 9.0).
  • a microwave oven microwave oven
  • CSF1R molecule expression was observed in 78 cases / 100 cases, and WFA epitope expression was observed in 76 cases / 100 cases. Met. In 70/100 cases, co-expression of CSF1R molecule and WFA epitope was observed.
  • Example 2 Selection of clinical trial patients (LC (+) HCC (-)) suffering from cirrhosis (LC) and not suffering from hepatocellular carcinoma (HCC) from January 1998 to January 2013
  • sera obtained from 214 patients with chronic liver disease type C who visited Nagoya City University Hospital were used.
  • HBs antigen positive patients and patients who developed malignant diseases of other organs within 3 months from the time of entry were excluded.
  • the median observation period was 60 months (1 to 195 months), and patients with Child Pew classification C were excluded from the study because the cancer rate and prognosis could not be accurately assessed due to transfer.
  • Liver fibrosis was evaluated by liver biopsy tissue, ultrasound, or CT.
  • Hepatocellular carcinoma was diagnosed by histological examination or diagnostic imaging based on the criteria of the American Society of Liver Disease. The fibrosis stage was evaluated individually by two pathologists using METAVIR, and cirrhosis was defined as F4. This study was approved by the Nagoya City University Hospital Ethics Committee based on the 1975 Declaration of Helsinki, with written consent. Patient selection is shown in FIG. 214 patients (chronic hepatitis [CH] 99 patients, cirrhosis [LC] 115 patients) were entered, and among the 115 LC patients, 59 (HCC-LC) had hepatocellular carcinoma. Eventually, 27 patients with poor liver cancer control (23 patients) or 3 cm ⁇ 3 or more hepatocellular carcinomas were excluded.
  • Example 3 Indicators for predicting the risk of developing hepatocellular carcinoma in patients with cirrhosis (LC) In this example, examination of indicators for predicting the risk of developing hepatocellular carcinoma among patients with cirrhosis (LC) I do.
  • WFA + -CSF1R% was promising as a predictor (HR 4.06, 95% CI 1.63-10.13, p ⁇ 0.001).
  • HCC hepatocellular carcinoma
  • LC (+) HCC (-) cirrhosis
  • WFA + -CSF1R% cutoff value examined above
  • Example 4 Prognosis (survival rate) of cirrhosis patients (LC (+) HCC (-)) without hepatocellular carcinoma (4-1) Survival rate by WFA + -CSF1R value in all patients WFA + -CSF1R Since the value increases in the progression of fibrosis, WFA + -CSF1R was evaluated as a predictor of LC patients. Clinical and cancer-related factors were assessed by time-dependent ROC curve AUC (area under the 95% confidence interval ROC curve during the total observation period) and risk calculated by Cox regression analysis.
  • AUC area under the 95% confidence interval ROC curve during the total observation period
  • the value showing the minimum P value in Cox regression analysis was determined as the optimum cutoff value of WFA + -CSF1R (310 ng / ml, FIG. 8).
  • WFA + -CSF1R viability WFA + -CSF1R value by value to increase as the progress of fibrosis was evaluated whether can become a WFA + -CSF1R the prognostic factors of LC patients.
  • WFA + -CSF1R value it is demonstrated that it is excellent as prognosis (survival) factor in LC patients, the value of the WFA + -CSF1R calculated in the minimum P value method, the prognosis of LC patients Functions as a forecast index.
  • Example 5 Preparation of standard CSF1R glycoprotein (5-1) Construction and purification of rCSF1R expression system
  • a rapid microassay kit for WFA + -CSF1R developed as a serum glycoprotein marker for cirrhosis
  • the production system of the glycoprotein standard product was examined. Since the measurement kit is an antibody-lectin sandwich detection system, the glycoprotein standard requires an epitope that reacts with each of the antibody and the lectin.
  • selection of cells producing a glycoprotein having a target sugar chain is considered to be very important.
  • the expression of a glycoprotein having a sugar chain that binds to WFA has already been established in the expression of liver fibrosis marker WFA + -M2BP.
  • CSF1R protein is a membrane protein consisting of 972 amino acids (SEQ ID NOs: 1, 2), 1-19 amino acids are signal sequences, 20-517 amino acids are extracellular regions, 518-538 amino acids are transmembrane regions, and 539-972 amino acids are It is an intracellular region. There are 11 consensus sequences for N-linked sugar chain addition in the extracellular region, and it is considered that N-linked sugar chains are bound to all or part of them. Based on this information, the recombinant CSF1R (rCSF1R) amplified its own signal sequence and a region encoding the extracellular region of 1-489 amino acids by PCR and introduced it into an expression vector.
  • rCSF1R recombinant CSF1R
  • human monocytic leukemia cell line (THP-1) -derived cDNA as a template, two primers, Fwd: 5'- AGGCCATGGGCCCAGGAGTTCTGCTGCT -3 ', (SEQ ID NO: 5) Rev: 5'-g gaattc GTTGTGGGCCCTGCACTCGTAG -3 '(underlined EcoRI site) (SEQ ID NO: 6)
  • the amplified 1.5 Kbp DNA fragment was subcloned into pCRII-Blunt vector (Invitrogen), and the amplified nucleic acid sequence was confirmed with Genetic Analyzer 3130xl (Applied Biosystems).
  • the DNA fragment cleaved with EcoRI was inserted into the EcoRI site before the DDDDK tag sequence of the expression vector pcDNA3.1neo (+) DDDDK (modified from Invitrogen) to construct pcDNA3.1-CSF1R-tag.
  • rCSF1R expressed from this vector has a DDDDK tag sequence at the C-terminus.
  • This plasmid was transfected into human fetal kidney-derived cell line HEK293 cells using Lipofectamine LTX (Invitrogen), and 1 mg / mL G418 (Nacalai Tesque) was added to the medium to select stable expression lines.
  • the culture supernatant after culturing the constructed stable expression strain in DMEM + 10% FCS + PS medium in a confluent state for 48 hours was repeatedly collected 3 times, and the supernatant was collected after centrifugation at 3100 rpm for 10 minutes.
  • the rCSF1R protein was purified from the collected culture supernatant using an anti-DDDDK antibody column (MBL).
  • MBL anti-DDDDK antibody column
  • the culture supernatant filtered through a 0.45 ⁇ m filter was applied to a DDDDK antibody column, and the passed supernatant was applied to the column again. Washing was performed with PBS buffer solution containing 0.1% Tween, 10 times the volume of the antibody column, and further with PBS buffer solution.
  • rCSF1R Elution of rCSF1R bound to the antibody column with PBS buffer containing 5 times the volume of the antibody column DDDDK peptide, and removal of the DDDDK peptide used for elution using an ultrafiltration membrane (Amicon 10K) Protein concentration was performed.
  • the rCSF1R protein obtained in the present invention is hereinafter also referred to as “standard CSF1R (protein)”.
  • the purified rCSF1R was stored at ⁇ 30 ° C. after measuring the protein concentration.
  • the lectin microphone alloarray used was one in which 45 different lectins were immobilized at 3 spots each (LecChip TM , Glyco Technica Co., Ltd.). 200 ng / well of LDN-positive and LDN-negative recombinant CSF1R diluted with a buffer solution was applied to the above array, and a lectin binding reaction was performed at 20 ° C. for 12 hours while gently shaking. After the reaction, human IgG was added at 2 ⁇ g / well and blocked for 30 minutes.
  • the sample solution containing the blocking agent on the array was removed, washed with a dedicated buffer three times, and then biotinylated anti-CSF1R polyclonal antibody (R & D Systems) diluted 100 times with a buffer containing 20 ug / mL human IgG. ) And the antibody was allowed to bind for 1 hour at 20 ° C. with gentle shaking. After the reaction, remove the antibody solution and wash 3 times with the dedicated buffer. Then add Cy3-conjugated streptavidin (GE Healthcare) diluted 5000 times with the buffer solution, and gently shake at 20 ° C for 1 hour. The next reaction was performed.
  • the secondary reaction solution was removed and washed with a dedicated buffer three times, and then the signal intensity was measured using a scanner for lectin microarray (GlycoStation TM Reader 1200) manufactured by Glyco Technica Co., Ltd. After calculating the true value by subtracting the background value, the average value between the three spots of each lectin was calculated, the maximum signal intensity of all lectins was determined as the reference value, the relative value was obtained, and the quantification was performed (FIG. 10). ).
  • N-linked glycosylation sites are determined by the IGOT method, and each N-linked glycosylation site is determined by the GlycoRidge method. Structural analysis was performed.
  • the GlycoRidge method is a technique for analyzing the peptide sequence and sugar chain structure of the recombinant protein developed by the present inventors (Noro E, et al, J Proteome Res. 2015 Sep 4; 14 (9): 3823-34. ) And generally follow the procedure below.
  • Recombinant glycoprotein is reductively alkylated with DTT and iodoacetamide, and after digestion with trypsin, the recovered glycoprotein is heated at high temperature (for example, 80 ° C., 2 hours) under acidic conditions (pH 2 or lower) to excise sialic acid.
  • the glycopeptide is analyzed by LC / MS method and the exact mass of each glycopeptide signal is listed.
  • the monosaccharide composition of the sugar chain portion is estimated from the calculated mass of the peptide containing the glycosylation site and the mass difference between the observed glycopeptides, and the addition site presumed to contain the estimated sugar chain motif is identified.
  • the glycan released from the glycopeptide is analyzed by MALDI-TOF MS, and if a fragment ion corresponding to the putative glycan motif is detected from the glycan having a composition that seems to contain the putative glycan motif, It can be confirmed that the addition position (peptide sequence) was correct.
  • the procedure was as follows. Recombinant CSF1R is subjected to reductive alkylation (reacted with DTT of equal weight of protein and iodoacetamide (protein ⁇ 2.5 times weight) and then dialyzed) followed by trypsin digestion and amide 80 column ( Tosoh Corporation: TOSOH) recovered the glycopeptide. This was heated under acidic conditions (pH ⁇ 2) at 80 ° C. for 2 hours to excise sialic acid. This glycopeptide was analyzed by LC / MS method, and the exact mass of each glycopeptide signal was listed (error is 2 ppm or less: Thermo Scientific LTQ-Orbitrap Velos).
  • the glycan released from the glycopeptide is analyzed by MALDI-TOF MS, and the HexNAc-HexNAc fragment ion is detected from the glycan of the composition that seems to contain LacDiNAc (hereinafter also referred to as LDN glycan) and its presence is confirmed. did.
  • Example 6 Confirmation of LDN sugar chain position on recombinant CSF1R using LDN-deficient strain (6-1) Construction of LDN-deficient strain Production of rCSF1R obtained in (5-1) of (Example 5) CRISPR / Cas9 system (Jennifer A. Doudna, et al., Science 28 Nov 2014), which encodes B4GALNT3 and B4GALNT4, which are glycosyltransferases having LDN sugar chain-specific transfer activity for transformed HEK293 cells : Vol.
  • an LDN-deficient strain was prepared by introducing an inactive mutation, and the standard CSF1R sugar lacking the LDN sugar chain (the sugar chain to which WFA binds) Protein was produced. Specifically, in HEK293 cells expressing LDN, mutations were successively introduced into the B4GALNT3 and B4GALNT4 genes using Invitrogen's GeneArt CRISPR Nuclease Vector Kit.
  • a plasmid was constructed by cloning the target sequence shown in exon2 (SEQ ID NO: 7) into GeneArt CRISPR Nuclease CD4 Vector, and transferred to HEK293 cells using Lipofectamine LTX (Invitrogen). Erected. After 24-48 hours, the plasmid-introduced cells were selected using CD4 Enrichment Kit (Invitrogen), and a plurality of single clones were isolated by limiting dilution.
  • the sequence around the target site is amplified by PCR with the primer set consisting of the base sequences shown in (SEQ ID NO: 8) and (SEQ ID NO: 9) from the genomic DNA of the isolated single clone strain, and the base is decoded to comprise 3000 bp It was confirmed that a frameshift mutation due to adenine insertion was introduced at position 209 of the coding region of the B4GALNT3 gene.
  • a plasmid was constructed by cloning the target sequence shown in exon2 (SEQ ID NO: 10) into GeneArt CRISPR Nuclease CD4 Vector, and Lipofectamine LTX was used to construct B4GALNT3 mutant cells. Transfected.
  • the sequence around the target site was amplified by PCR with a primer set consisting of the base sequences shown in (SEQ ID NO: 11) and (SEQ ID NO: 12), and the base was decoded. It was confirmed that a frameshift mutation due to deletion of cytosine at position 184 in the coding region of the B4GALNT4 gene consisting of 3120 bp was introduced.
  • Example 7 Preparation of anti-CSF1R antibody (7-1) Immunization of mice with CSF1R protein
  • Commercially available recombinant CSF1R protein (R & D Systems Inc. 329-MR-100) was used in mice (Balb / c mice, 8 weeks old). The female was immunized.
  • CSF1R protein dissolved in physiological saline is mixed with complete Freund's adjuvant, and this is mixed with 50 ⁇ g for the first day (Day 0), 25 ⁇ g for Day 14, 25 ⁇ g for Day 29, 25 ⁇ g for Day 42, Day 66 was immunized by intraperitoneal injection of 10 ⁇ g.
  • Orbital blood sampling was performed on mice periodically, and immunization was performed while monitoring the increase in antibody titer against the antigen in the serum.
  • Antibody-producing cells were collected from immunized mice that were confirmed to have sufficiently increased antibody titers. Since the collection was preferably 2 to 5 days after the last immunization, it was collected 3 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, and spleen cells or local lymph node cells are preferred.
  • a method for collecting antibody-producing cells from mice may be performed according to a technique known in the art. Therefore, spleen cells were collected and a fusion operation described later was performed.
  • a hybridoma producing an anti-CSF1R monoclonal antibody can be produced by cell fusion of antibody-producing cells and myeloma (myeloma) cells.
  • myeloma myeloma
  • P3U1 cells immunized mouse-derived spleen cells and mouse myeloma cells (P3U1 cells), in accordance with the usual method (described later), each cell is washed with RPMI medium and then mixed, and cell fusion with a cell fusion promoter (PEG1500) Went.
  • PEG1500 cell fusion promoter
  • HAT medium RPMI1640 medium with 100 units / mL penicillin, 100 ⁇ g / mL streptomycin and 10% fetal bovine serum (FBS), 10-4 M hypoxanthine, 1.5 ⁇ 10 ⁇ 5 M thymidine and 4 ⁇ after cell fusion.
  • FBS fetal bovine serum
  • the cells were cultured in a medium supplemented with 10 ⁇ 7 M aminopterin) and selectively cultured so that only the fused cells survived.
  • Monoclonal cells were then obtained by a limiting dilution method in order to select cells that grow after about 10 days from the start of culture in a selective medium as hybridomas.
  • the cell solution (concentration) was seeded in a 96-well culture plate so as to prepare a dilution series from a thicker one to a thinner one, and a limited number of cell-derived hybridoma cell groups were selected, A clone that produced an antibody against CSF1R (including positive wells of a 96-well plate) was selected by screening described below.
  • the screening method is as follows. It was screened by enzyme immunoassay (ELISA method) whether or not the target anti-CSF1R monoclonal antibody was contained in the culture supernatant of the hybridoma that had proliferated. A part of the culture supernatant contained in the well in which the hybridoma was cultured was collected, and the binding activity to the CSF1R recombinant protein used as an immunogen was used as an index. CSF1R recombinant protein was immobilized on a 96-well plate (100 ⁇ L / well at 1 ⁇ g / mL), blocked, and 100 ⁇ L of culture supernatant was added and reacted at 37 ° C. for 1 hour.
  • ELISA method enzyme immunoassay
  • Positive clones were selected by ELISA screening and limiting dilution (specifically, seeded at a concentration containing about 0.3 cells per well in a 96-well culture plate). There were 205 positive wells at the time of the primary screening, which were expanded and further narrowed down, 100 wells at the time of the secondary screening, and finally 33 clones of hybridomas that were anti-CSF1R monoclonal antibody producing cells were selected. The 33 clones of anti-CSF1R monoclonal antibody-producing hybridomas finally selected by the screening method were named CSR-1 to CSR-33, respectively (Table 5).
  • the culture supernatant of the obtained hybridoma cells was adjusted to about 100 to 1 L. This was purified by affinity chromatography using a column on which protein G was immobilized.
  • hybridomas producing typical types of anti-CSF1R monoclonal antibodies CSR-3, CSR-4, CSR-18, CSR-21 and CSR-30 are CSR-3. (Accession number: NITE BP-02117) or CSR-4 (Accession number: NITE BP-02118) CSR-18 (Accession number: NITE BP-02119) CSR-21 (Accession number: NITE BP-02120) CSR-30 (Deposit number: NITE BP-02121), deposited on September 10, 2015 at the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (NPMD), and then on September 7, 2016 It was transferred to the deposit.
  • NITE BP-02117 or CSR-4 (Accession number: NITE BP-02118)
  • CSR-18 Accession number: NITE BP-02119
  • CSR-21 Accession number: NITE BP-02120
  • CSR-30 Deposit number: NITE BP-02121
  • hybridoma cell lines can be suitably cultured at 37 ° C. using a medium in which 10% FBS is added to RPMI1640.
  • the anti-CSF1R monoclonal antibody can be recovered by a conventional technique.
  • purification of the antibody is required, ion exchange chromatography, affinity chromatography using protein A or protein G, gel chromatography, It can refine
  • Example 8 Performance evaluation of each anti-CSF1R antibody (8-1) Biochemical analysis by Western blotting
  • the anti-CSF1R antibody was used to detect the molecule by Western blotting.
  • the Western blot method followed a general method. First, as shown in FIG. 14 (left), CSF1R (M-CSFR) and other samples were electrophoresed using 10% polyacrylamide gel under SDS-PAGE reducing conditions and transferred to a PVDF membrane. After blocking with PBS containing 5% skim milk, the mixture was reacted with a primary antibody (each clone of anti-CSF1R antibody) at room temperature for 1 hour.
  • a primary antibody each clone of anti-CSF1R antibody
  • PVDF membrane After washing the PVDF membrane, it was reacted with a secondary antibody (0.5 ⁇ g / mL HRP-labeled anti-mouse IgG antibody) at room temperature for 1 hour. These PVDF membranes were washed and detected by chemiluminescence using a Western blotting detection reagent (Perkin Elmer).
  • a secondary antibody 0.5 ⁇ g / mL HRP-labeled anti-mouse IgG antibody
  • each standard glycoprotein sample was electrophoresed on a 10% polyacrylamide gel under SDS-PAGE reducing conditions and transferred to a PVDF membrane. After blocking with PBS containing 5% skim milk, the mixture was reacted with the primary antibody (each monoclonal antibody clone of anti-CSF1R antibody) at room temperature for 1 hour.
  • Antibody-antibody ELISA measurement system using anti-CSF1R antibody obtained in the present invention (9-1) Antibody-antibody Detection of total CSF1R molecule by ELISA measurement system Using anti-CSF1R monoclonal antibody, Molecules (total CSF1R molecules) were detected by an antibody-antibody ELISA measurement system. Each established anti-CSF1R monoclonal antibody was immobilized on an ELISA plate, and a commercially available anti-CSF1R polyclonal antibody was used on the detection side to examine whether it could be used in an ELISA measurement system.
  • the combination of antibodies may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and a detection system is constructed with a combination of antibodies with high sensitivity.
  • a detection system is constructed with a combination of high sensitivity and low background noise.
  • Example 10 Antibody-WFA lectin using anti-CSF1R antibody obtained in the present invention
  • Sandwich ELISA measurement system (10-1) method anti-CSF1R monoclonal antibody is used and antibody-WFA lectin of the molecule is used. Detection was performed by a sandwich ELISA measurement system.
  • the anti-CSF1R monoclonal antibody was used on the ELISA plate immobilization side, while the feasibility of use in an antibody-lectin sandwich ELISA measurement system using WFA lectin on the detection side was examined.
  • the antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. Used on the phase side) in a sandwich detection system.
  • a detection system is constructed with a combination of high sensitivity and low background noise.
  • WFA lectins may be used, or recombinant WFA, particularly LDN-specific monomeric recombinant WFA (srWFA) may be used.
  • srWFA LDN-specific monomeric recombinant WFA
  • Each antibody was diluted with PBS to 4 ⁇ g / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 ⁇ L / well for blocking.
  • HRP horseradish peroxidase
  • Jackson horseradish peroxidase
  • FIG. 21 (left is srWFA, right is nWFA) is a sandwich ELISA system in which LDN sugar chain-specific monomeric recombinant WFA (srWFA) or commercially available natural WFA (nWFA) is combined with an anti-CSF1R antibody.
  • srWFA LDN sugar chain-specific monomeric recombinant WFA
  • nWFA commercially available natural WFA
  • FIG. 21 (left is srWFA, right is nWFA) is a sandwich ELISA system in which LDN sugar chain-specific monomeric recombinant WFA (srWFA) or commercially available natural WFA (nWFA) is combined with an anti-CSF1R antibody.
  • srWFA LDN sugar chain-specific monomeric recombinant WFA
  • nWFA commercially available natural WFA
  • Example 11 Detection of WFA + -CSF1R molecule by antibody- WFA lectin sandwich ELISA measurement system (Example 8) and detection of the molecule by antibody- WFA lectin sandwich ELISA measurement system Went.
  • the sandwich ELISA assay system was examined using anti-CSF1R antibodies on the ELISA plate immobilization side and the detection side, respectively.
  • the antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. (Used on the phase side) and performed in a sandwich detection system.
  • WFA lectins commercially available natural WFA and monomeric recombinant WFA (srWFA) were used.
  • each antibody was diluted with PBS to 4 ⁇ g / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 ⁇ L / well for blocking. After discarding and washing the blocking solution, the sample (Example 5) has a recombinant CSF1R having LDN sugar chains prepared in HEK293 cells, and (Example 6) has an LDN sugar chain prepared in sugar chain gene knockout cells.
  • HRP horseradish peroxidase
  • Jackson horseradish peroxidase
  • the anti-CSF1R antibodies CSR-3, CSR-4, CSR-18, CSR-21, and CSR-30 prepared in (Example 8) can be combined with either monomeric srWFA or nWFA. It was shown that the WFA / VVA-binding sugar chain present above can be detected (FIG. 22). In particular, it was shown that the LDN-specific monomer srWFA can specifically identify the presence or absence of LDN sugar chains (upper part of FIG. 22). It should be noted that nWFA has a reactivity with rCSF1R that does not have a LDN sugar chain slightly, and is considered to react with sugar chains other than LDN.
  • Example 12 Antibody-WFA lectin sandwich using antibody CRS-3, and anti-CSF1R antibody (CSR-3) and WFA lectin prepared by detection of CSF1R molecule by antibody-antibody sandwich ELISA measurement system (Example 8) , And rCSF1R (LDN +) and rCSF1R adjusted to the same concentration (by dilution series) using CSR-3-WFA lectin sandwich ELISA and CSR-3-commercial antibody sandwich ELISA measurement system using commercially available antibodies (R & D Systems) (LDN-) was detected. Specifically, each antibody was diluted with PBS to 4 ⁇ g / mL and added to an ELISA microplate at 100 uL / well.
  • biotin-labeled WFA lectin or monomeric recombinant WFA: srWFA
  • biotin-labeled anti-CSF1R antibody R & D biotinylated anti-CSF1R pAb Cat # BAF329
  • HRP horseradish peroxidase
  • Jackson horseradish peroxidase
  • Example 13 Detection of marker molecule by antibody-VVA lectin sandwich ELISA measurement system
  • LacdiNAc / GalNAc binding lectin such as VVA lectin can also be used. Therefore, the anti-CSF1R antibody was used to detect the molecule by an antibody-VVA lectin sandwich ELISA measurement system.
  • the sandwich ELISA assay system was examined using anti-CSF1R antibodies on the ELISA plate immobilization side and the detection side, respectively.
  • the antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. Used on the phase side) in a sandwich detection system.
  • a detection system is constructed with a combination of high sensitivity and low background noise.
  • Each antibody was diluted with PBS to 4 ⁇ g / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 ⁇ L / well for blocking.
  • the sample (Example 5) has a recombinant CSF1R having LDN sugar chains prepared in HEK293 cells, and (Example 6) has an LDN sugar chain prepared in sugar chain gene knockout cells.
  • Example 14 Detection of WFA + -CSF1R with anti- CSF1R antibody of the present invention (14-1) Detection with WFA lectin-anti-CSF1R antibody sandwich ELISA system
  • the anti-CSF1R monoclonal prepared in (Example 8) In order to confirm that the antibody can be used for detection of liver disease marker molecule WFA + -CSF1R, it was applied to anti-CSF1R antibody-WFA lectin sandwich ELISA according to the method described in Non-Patent Document 2 and WFA + -CSF1R values were measured.
  • serum samples from healthy subjects (17 subjects, NHS), HBV-infected hepatocellular carcinoma patient pool serum (K1), HCV-infected liver cancer patient pool serum (K2), HCV infection Hepatocellular carcinoma patient (splenectomized) pooled serum (K3) was used.
  • HBV-infected hepatocellular carcinoma patient pool serum K1
  • HCV-infected liver cancer patient pool serum K2
  • HCV infection Hepatocellular carcinoma patient splenectomized pooled serum
  • VVA lectin exhibits a binding ability to glycans on CSF1R that is specifically increased in patients with hepatocellular carcinoma, inferior to WFA lectin even when using actual clinical body fluid samples.
  • VVA lectin in an assay system in combination with an anti-CSF1R antibody it means that “the amount of CSF1R containing a WFA / VVA lectin-binding sugar chain” in a clinical body fluid sample can be measured.
  • Example 15 Construction of "CSF1R-specific glycan-binding lectin-anti-CSF1R antibody sandwich ELISA" system that can be used as an ELISA for measuring "total CSF1R amount”
  • hepatocellular carcinoma of the present invention is used.
  • the measurement of the onset risk index “WFA + -CSF1R%”, that is, the ratio of “CSF1R amount having WFA / VVA-binding sugar chain” to “total CSF1R amount” in the test sample is “CSF1R-specific lectin binding” It is demonstrated that “the amount of CSF1R having a WFA / VVA-binding sugar chain” relative to “the amount of CSF1R having a functional sugar chain” may be measured.
  • a measurement system is constructed to demonstrate that the lectin-antibody sandwich assay system can be used in place of a sandwich measurement system that uses two anti-CSF1R antibodies for total CSF1R measurement.
  • sugar chain reactivity (binding property) is different for each lectin, a value (ratio) relative to the WFA (or VVA) -CSF1R value in the determination step for comparing the measured values in this example. What was calculated as was used.
  • CSR-3 anti-CSF1R antibody
  • Example 8 Nunc Immobilizer Amino plate (Thermo Scientific Hook, 43613) was coated with anti-CSF1R antibody (CSR-3) prepared at 4 ⁇ g / mL (Example 8) for 2 hours and washed buffer (PBS buffer containing 0.05% Tween20, pH 7.4) ) And then blocked with TBS (50 mM Tris-pH 8.0, 0.15 M NaCl) at 4 ° C. overnight.
  • recombinant CSF1R (LDN +) of (Example 5) and recombinant CSF1R (LDN-) of (Example 6) were used as substitutes for CSF1R derived from diseased samples and normal samples, respectively.
  • biotin-binding lectins diluted with washing buffer 250-20,000 times dilution
  • WFA Vector Laboratory, B-1355, 5,000 times dilution
  • VVA Vector Laboratory, B-1235, diluted 250 times
  • RCA120 Vector Laboratory, B-1085, diluted 20,000 times
  • DSA Vector Laboratory, B-1185, diluted 10,000 times
  • PHA-E4 J-Oil Mills, J211, 2,000 times diluted) Dilution
  • SNA Vector Laboratory, B-1305, 10,000 times dilution
  • SSA J-Oil Mills, J218, 3,000 times dilution
  • TJA-1 Seikagaku, 300443, 500 times dilution
  • LEL Vector Laboratory
  • B-1175 5,000-fold dilution
  • STL Vector Laboratory, B-1165, 1,000-fold dilution
  • Con A Vector Laboratory, B-1005, 20,000-fold dilution
  • Con A Vector Laboratory, B-1005, 20,000-fold dilution
  • the signal value when anti-CSF1R antibody is used as a detection probe in the above ELISA is the total CSF1R measurement value, and the values of recombinant CSF1R [LacdiNAc (LDN) sugar chain (+)] and [LDN sugar chain (-)] The concentration was adjusted to match.
  • the total CSF1R measured values after concentration adjustment agreed well with both the BSA diluted solution and the 10% NHS diluted solution (FIGS. 28A and 28B). This indicates that this measurement system can be used without any problems in either the BSA dilution system or the 10% NHS dilution system.
  • rCSF1R LDN +
  • rCSF1R LDN +
  • rCSF1R LDN-
  • the ratio of rCSF1R (LDN +) to rCSF1R (LDN-) was higher for anti-CSF1R antibodies.
  • lectin it was 2.0 ⁇ 0.25 (1.7 to 2.6) compared to 2.2. Therefore, any of the lectins of RCA120, DSA, PHA-E4, SNA, SSA, TJA-1, LEL, STL, and Con A can be used to measure the total CSF1R value as a substitute for the anti-CSF1R antibody.
  • the concentration of the CSF1R molecule to be measured was corrected and compared.
  • the correction method is as follows.
  • the rCSF1R (LDN +) protein has an LDN sugar chain as shown in FIG. 13 described above, but the positive rate of the LDN sugar chain is about 60% of all proteins. Therefore, the correction of the rCSF1R (LDN +) concentration was calculated as a coefficient of 0.6 and used as a correction value.
  • rCSF1R (LDN +) correction value create a standard curve, apply the rCSF1R (LDN-) signal value to this to calculate the correction value (corrected relative concentration value), and rCSF1R (LDN +) correction that can be compared with each other This was graphed as a value, rCSF1R (LDN-) correction value (FIG. 30). Similarly, the results with this correction value are similar to those obtained with the anti-CSF1R antibody for all lectins, and the value of rCSF1R (LDN +) is higher than that of rCSF1R (LDN-).
  • the detection system (antibody-lectin sandwich ELISA system) constructed in the above (15-1) to (15-4) can be measured without problems even in serum.
  • the molecular weight of a disease-specific sugar chain can be determined by a multi-lectin assay that combines a disease-specific probe (lectin) such as WFA or VVA with a probe that binds to the CSF1R common sugar chain (lectin). It has been demonstrated that by measuring it is possible to determine whether or not the person is afflicted with the disease.
  • CSF1R protein target molecule
  • Such a measurement method using a multi-lectin assay can be performed by forming a complex in which a CSF1R protein separated and purified from a clinical sample is sandwiched with one or more lectins, and detecting (quantifying) this complex. I can do it. About the detection of the complex of CSF1R protein and a lectin, it can detect with the sandwich ELISA system currently performed in this application.
  • a disease-specific probe ie, WFA or VVA lectin
  • a probe having binding ability to a CSF1R-specific common sugar chain RCA120, DSA, PHA-E4
  • the CSF1R protein separated and purified from the clinical sample is added and reacted, and the other is liquid side
  • a (multi) lectin-protein complex may be formed, and the amount of this complex may be detected, or a system using a lectin-lectin sandwich detection system may be used.
  • the complex of CSF1R protein and lectin can be detected by capillary electrophoresis or separation / detection system using microfluidics technology.
  • enzyme immunoassay two-antibody sandwich ELISA, gold colloid method, radioimmunoassay, latex agglutination immunoassay, fluorescence immunoassay, western blotting, immunohistochemistry, surface plasmon resonance (SPR method) ) Or a qualitative or quantitative method using a quartz crystal microbalance (QCM) method or the like.

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Abstract

The present invention provides a method and a kit for accurately predicting the prognosis (mortality rate) and risk of developing hepatocellular carcinoma in liver cirrhosis patients. The present invention provides: a method by which an "index for evaluating the risk of developing hepatocellular carcinoma," which is used for predicting the prognosis and risk of developing hepatocellular carcinoma in liver cirrhosis patients, is calculated as the ratio of CSF1R that contains WFA/VVA-binding sugars relative to the total CSF1R content of bodily fluid (blood serum) (WFA+–CSF1R%); and a method by which a "prognosis evaluation index" is calculated as the amount of CSF1R that contains WFA/VVA-binding sugars (WFA+–CSF1R ng/mL). Furthermore, an optimal cutoff value was determined for each of the indices, and it was proven that: the risk of developing hepatocellular carcinoma was significantly high when the "index for evaluating the risk of developing hepatocellular carcinoma" in a subject was equal to or greater than the optimal cutoff value; and the prognosis was significantly poor when the "prognosis evaluation index" was equal to or greater than the optimal cutoff value. In addition, anti-CSF1R antibodies (CSR-1–30), which are exceptional for detecting CSF1Rs such as CSF1Rs that contain WFA/VVA-binding sugars in a bodily fluid sample, were provided. It was discovered that srWFA and VVA lectins other than WFA lectins can be used, and it was additionally proven that measuring the amount of CSF1R that binds to a CSF1R-specific lectin is preferable to measuring the total amount of CSF1R. It was also possible to provide, inter alia, a kit for measuring the "prognosis evaluation index" and/or "index for evaluating the risk of developing hepatocellular carcinoma" in a liver cirrhosis patient, the kit including these anti-CSF1R antibodies and lectins as constituent elements.

Description

肝硬変患者における肝細胞がん発生リスク及び予後を予測するための方法Methods for predicting hepatocellular carcinoma risk and prognosis in patients with cirrhosis
　本願発明は、重篤な肝疾患病態である肝硬変において、肝細胞がんへの進行を正確に把握し、その予後及び治療後の再発を評価するための方法及びキットに関する。より詳しくは、肝硬変（F4）における肝細胞がんの発生と相関性の高い肝細胞がん糖鎖バイオマーカーを利用して肝硬変又は肝細胞がんの重篤度を「肝細胞がんリスク判定指数」及び／又は「肝硬変予後判定指数」として定量化し、肝硬変における肝細胞がん発生リスクの判定、及び治療後の予後（生存率）を正確に判定するための方法及びキットを提供する。 The present invention relates to a method and kit for accurately grasping the progression to hepatocellular carcinoma and evaluating the prognosis and recurrence after treatment in cirrhosis, which is a serious liver disease state. More specifically, the severity of liver cirrhosis or hepatocellular carcinoma was determined using a hepatocellular carcinoma sugar chain biomarker highly correlated with the occurrence of hepatocellular carcinoma in cirrhosis (F4). Provided are a method and a kit for quantifying as an “index” and / or “cirrhosis prognosis determination index” to determine the risk of developing hepatocellular carcinoma in cirrhosis and to accurately determine the prognosis (survival rate) after treatment.
　肝臓がんは、肝臓で発生する原発性肝がんと、転移性肝がんに大きく分けることができ、原発性肝がんの90％が肝細胞がん（Hepatocellular carcinoma：HCC）であるといわれている。
　肝細胞がん患者は、基礎疾患として、C型肝炎ウイルス、又はB型肝炎ウイルスに感染している場合が多く、ウイルス性肝炎に罹患した後、急性ウイルス性肝炎から、慢性ウイルス性肝炎、肝硬変へと徐々に病態の進行に伴って肝機能が低下し、肝炎病態の進行・持続とともに肝臓の線維化が進み、やがて肝硬変に至る。発がん率も病態の進行に伴い上昇し、慢性肝炎軽度（F1）又は中度（F2）では年率0.8～0.9％程度であるが、慢性肝炎重度（F3）になると年率3.5％になり、肝硬変（F4）からがんとなる確率は、年率7％にも上昇する。あるいは重篤な場合には肝不全を起こし、死に至ることもある。
　肝細胞がんの治療においても、早期のがん発見が治療、術後予後に大きく影響しているため重要であり、肝硬変患者の場合は、3ヶ月に1度程度の肝がん検出の検査を受ける必要がある。この検査をより簡便化するために、血液検査で正確にかつ簡便に発がんの有無が判定できる方法の提供が求められていた。 Liver cancer can be broadly divided into primary liver cancer that occurs in the liver and metastatic liver cancer. 90% of primary liver cancer is hepatocellular carcinoma (HCC). It is said.
Patients with hepatocellular carcinoma are often infected with hepatitis C virus or hepatitis B virus as the underlying disease. After suffering from viral hepatitis, acute viral hepatitis, chronic viral hepatitis, cirrhosis As the disease progresses gradually, the liver function decreases, and as the hepatitis condition progresses and continues, the fibrosis of the liver progresses, eventually leading to cirrhosis. The carcinogenic rate also increases as the disease progresses, and the annual rate is about 0.8 to 0.9% for chronic hepatitis mild (F1) or moderate (F2). However, when chronic hepatitis is severe (F3), the annual rate is 3.5%, and cirrhosis ( The probability of developing cancer from F4) increases to an annual rate of 7%. In severe cases, it may cause liver failure and death.
Even in the treatment of hepatocellular carcinoma, early detection of cancer is important because it significantly affects treatment and postoperative prognosis. In patients with cirrhosis, tests for detection of liver cancer once every three months It is necessary to receive. In order to further simplify this test, it has been required to provide a method that can accurately and easily determine the presence or absence of carcinogenesis by a blood test.
　現在、血清中の肝がんマーカーとしては、AFP(α－フェトプロテイン）（特許文献１）及びPIVKA-II（protein induced by Vitamin K absence or antagonist-II）（特許文献２）などが用いられることがあるが、いずれも特異性、感度とも十分ではなく正確な判定には至っていない。そのため、肝細胞がん早期発見のための検診では、超音波検査、コンピューター断層撮影（CT）、核磁気共鳴画像法（MRI）など画像検査が主であり、肝がんマーカーの使用はあくまで補助的な役割に過ぎない。 Currently, AFP (α-fetoprotein) (Patent Document 1) and PIVKA-II (protein-induced by Vitamin K absence or antagonist-II) (Patent Document 2) are used as serum liver cancer markers. However, neither specificity nor sensitivity is sufficient, and accurate determination has not been reached. Therefore, for screening for early detection of hepatocellular carcinoma, imaging tests such as ultrasonography, computed tomography (CT), and nuclear magnetic resonance imaging (MRI) are mainly used, and the use of liver cancer markers is only an aid. It is only a role.
　本発明者らは、以前から血液など体液の検査でがん化を検出できるような肝疾患病態を区別できる糖鎖マーカー及び肝細胞がんマーカーの提供を目指し、血清中に存在する各種糖タンパク質上の糖鎖構造の変化に着目した研究開発を行ってきた。そして、肝炎患者血清において、従来から肝細胞がんマーカーとして用いられていたCSF1R（非特許文献１など）のタンパク質量はF1からF4とステージが進むにつれ徐々に高くなるが、CSF1R糖タンパク質上の糖鎖構造に着目するとWFAレクチン結合性糖鎖が、F1～F3ステージではほとんど発現せずF4ステージの肝硬変患者で有意に増大することを見いだした（特許文献３，非特許文献２）。データの一部に肝細胞がんの発生を反映している可能性が示唆された（特許文献３）が、肝硬変患者群では肝がんを患っているか否かでの有意差が見いだせなかった（非特許文献２）。すなわち、血清中のCSF1R上のWFA結合性糖鎖量は、肝臓の病態の重篤度の判別及び肝硬変の検出にはきわめて有効なマーカーとなることが示されたのに対し、期待されていた肝細胞がんの検出についての有効性は実証できなかった。
　このように、血清中のCSF1R糖タンパク質量自体がF1からF4へとステージが進展するに従って高くなるため、肝臓の線維化度を示す指標となる可能性があり、血清中のCSF1R上のWFA結合性糖鎖量の数値の増大は、肝硬変発生の検出には有効な指標となるとしても、いずれも正確に肝細胞がん発生を予測しているとはいえない。特に肝硬変患者に対しての早期の肝細胞がんの予測に適用することは困難であった。
　したがって、血液試料から測定される数値のみで正確に肝硬変患者にも適用可能な肝細胞がん発生の予測のための技術開発は急務であった。 The present inventors have been aiming to provide a sugar chain marker and a hepatocellular carcinoma marker that can distinguish liver disease pathologies that can detect canceration by testing body fluids such as blood, and various glycoproteins present in serum. We have conducted research and development focusing on the changes in the sugar chain structure above. In sera of hepatitis patients, the protein level of CSF1R (such as Non-patent Document 1), which has been used as a hepatocellular carcinoma marker, gradually increases from F1 to F4 as the stage progresses. Focusing on the sugar chain structure, it was found that WFA lectin-binding sugar chains are hardly expressed in the F1 to F3 stages and significantly increase in patients with cirrhosis in the F4 stage (Patent Document 3 and Non-Patent Document 2). The possibility of reflecting the occurrence of hepatocellular carcinoma was suggested in part of the data (Patent Document 3), but in the cirrhosis patient group, no significant difference was found depending on whether or not he had liver cancer. (Non-patent document 2). That is, the amount of WFA-binding sugar chains on CSF1R in serum was expected to be a very effective marker for discrimination of the severity of liver pathology and detection of cirrhosis. The effectiveness in detecting hepatocellular carcinoma could not be demonstrated.
In this way, the amount of CSF1R glycoprotein in the serum itself increases as the stage progresses from F1 to F4, so it may be an indicator of liver fibrosis, and WFA binding on CSF1R in serum Although an increase in the value of the amount of sex sugar chain is an effective index for detecting the occurrence of cirrhosis, none of them accurately predicts the occurrence of hepatocellular carcinoma. In particular, it was difficult to apply to the early prediction of hepatocellular carcinoma for patients with cirrhosis.
Therefore, there is an urgent need to develop a technology for predicting the occurrence of hepatocellular carcinoma that can be applied to patients with cirrhosis accurately using only the values measured from blood samples.
　また、肝疾患の病態が進行して肝硬変（F4）までに線維化が進んだ場合、病変部の切除を行う肝切除治療が一般的であるが、肝切除治療による5年生存率は、F1であれば80％のところ、肝硬変の場合は38％に過ぎない。このことから、肝硬変患者のフォローアップのためには、肝硬変患者の予後を早い段階でできるだけ正確に判定し術後方針を決定する必要がある。現在、血清中のアルブミン値が3.5以上であれば肝臓全体の機能低下がみられないと判定して、予後を予測しているが、この数値は必ずしも肝硬変患者、または肝疾患の病態が高いステージにある（F4）患者の予後を正確に反映するものではなく、最終的には術者（医師）の経験と勘に任されている状況であった。
　したがって、肝硬変患者の予後判定のより正確な指標となる糖鎖マーカーや数値など、有効な予後診断につながる判定方法に対しても強い要望があった。 In addition, when fibrosis progresses by cirrhosis (F4) due to progression of liver disease, hepatectomy is generally performed to remove the lesion, but the 5-year survival rate from hepatectomy is F1. If so, 80%, but only 38% for cirrhosis. Therefore, in order to follow up patients with cirrhosis, it is necessary to determine the prognosis of patients with cirrhosis as accurately as possible at an early stage and to determine the postoperative policy. At present, if the serum albumin level is 3.5 or more, it is judged that there is no decrease in the function of the whole liver, and the prognosis is predicted. (F4) does not accurately reflect the patient's prognosis, and ultimately the situation depends on the experience and intuition of the surgeon (doctor).
Therefore, there has been a strong demand for a determination method that leads to an effective prognosis diagnosis, such as a sugar chain marker or a numerical value that is a more accurate index for prognosis determination of cirrhosis patients.
特開平１０－２６６２２号JP-A-10-26622 特開平８－１８４５９４号JP-A-8-184594 国際公開２０１１－００７７６４（WO2011/007764）International Publication 2011-007764 (WO2011 / 007764) 国際公開２０１４－０９８１１２（WO2014/098112）International Publication 2014-098112 (WO2014 / 098112)
　本発明の課題は、肝硬変患者に対しても、体液（血液）検査で正確かつ簡便にがん化の可能性を予測可能な肝細胞がんリスク判定方法を提供するものであり、また、肝硬変患者の予後を正確かつ簡便に判定するための肝硬変予後判定方法を提供するものである。具体的には、肝硬変患者の体液例えば血清中のCSF1R上の糖鎖構造の変化のうち、肝細胞がんの発生及び／又は肝硬変の予後を直接的に反映するWFA結合性糖鎖への変化の割合を正確に定量化する方法を提供しようとするものである。 An object of the present invention is to provide a method for determining the risk of hepatocellular carcinoma that can accurately and easily predict the possibility of canceration by a body fluid (blood) test even for cirrhosis patients. The present invention provides a method for determining the prognosis of cirrhosis for accurately and simply determining the prognosis of a patient. Specifically, among the changes in the sugar chain structure on CSF1R in body fluids such as serum of cirrhotic patients, changes to WFA-binding sugar chains that directly reflect the occurrence of hepatocellular carcinoma and / or the prognosis of cirrhosis It is intended to provide a method for accurately quantifying the proportion of
　上述のように、本発明者らは、体液（血清）中のWFA結合性糖鎖含有CSF1R量の増大が、肝臓の病態の重篤化と高い関連性を有することを見いだしており、さらに肝細胞がんの発生を反映している可能性も想定していた（特許文献３）ので、まずは、抗CSF1R抗体及びWFAレクチンを用いたアッセイ系を構築し、この測定系を用いて、肝炎患者由来および肝がん患者由来の体液（血清）試料のWFA結合性糖鎖含有CSF1R量を測定し、肝細胞がんに罹患しているか否かでWFA結合性糖鎖含有CSF1R量に差異が出るかどうかを検討した。その結果、肝炎患者全体では有意差をもって肝細胞がんの検出に有効であることがわかった。しかし、WFA結合性糖鎖含有CSF1R量の増大は肝炎病態の重篤度、線維化度との関連性も高いことから、本来肝細胞がんリスクが高いために正確な診断が必要なはずの肝硬変患者など重篤患者において、肝細胞がんの発生の正確な予測判定ができないという不備があった。
　そこで、本発明者らは、肝硬変には罹患しているが肝細胞がんに罹患していない患者群（LC patients without HCC）に着目し、これら患者をフォローアップすると共に、その後の肝細胞がんの発生率と相関のあるマーカー及び計算式を詳細に検討した。
　その結果、体液（血清）中の全CSF1Rに対するWFA結合性糖鎖含有CSF1Rの割合（WFA+-CSF1R％）、同様にCSF1R特異的レクチン結合性糖鎖含有CSF1Rに対するWFA結合性糖鎖含有CSF1Rの割合が、肝硬変患者において、肝細胞がんの発生と高い相関関係にあることを見いだした。なお、ここで、「CSF1R特異的レクチン」とは、健常人と患者の血清など体液中のCSF1R上の糖鎖への反応性において差が無いものをいう。「CSF1R含有糖鎖特異的レクチン」「CSF1R特異的共通糖鎖結合性レクチン」「全CSF1R共通糖鎖結合性レクチン」「CSF1R恒常的糖鎖構造結合性レクチン」ともいう。つまり体液中の全CSF1Rタンパク質上の糖鎖に反応するレクチンを意味するから、体液中の全CSF1R量に対するWFA結合性糖鎖含有CSF1R量を測定する代わりに「CSF1R特異的レクチン結合性糖鎖量」に対する「WFA結合性糖鎖量」の割合と言い換えることもできる。各レクチン結合性糖鎖量は、各レクチンへの反応性の強さに比例するから、結局、肝硬変患者における肝細胞がんの発生率は、被検体液試料中の2種類のレクチンに対する反応性の比率のみで測定することができることを意味する。CSF1R特異的レクチンとしては、典型的には、非特許文献１，Fig3Bに示されたRCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA等のレクチンが相当する。後者は、実質的に前者と同様に体液中の全CSF1Rに対するWFA結合性糖鎖含有CSF1Rの割合を示すものなので、両者を併せて「肝細胞がんリスク判定指数（WFA+-CSF1R％）」と命名した。具体的には、「WFA結合性糖鎖含有CSF1R量／全CSF1R量×100」値又は「WFA結合性糖鎖含有CSF1R量／CSF1R特異的レクチン結合性糖鎖含有CSF1R量×100」値、もしくは「CSF1R分子上のWFA結合性糖鎖量／CSF1R特異的レクチン結合性糖鎖量×100」値である。これらの値と肝硬変患者の肝細胞がんの発がん率をログランク検定で決定した最小P値法により、最適カットオフ値を求めると35％となった。そこで、肝細胞がんを発症していない肝硬変患者を、35％以上の高値群と35％未満の低値群とに分けてカプラン・マイヤー解析を行うと、5年の累積発がん率はWFA+-CSF1R％高値群で有意に高い（P = 0.006又は0.005）という結果を得た。このことは、「WFA+-CSF1R％」の値が、肝硬変患者にとってきわめて有効な「肝細胞がんリスク判定指数」となることを示すものである。 As described above, the present inventors have found that an increase in the amount of CSF1R containing a WFA-binding sugar chain in a body fluid (serum) has a high correlation with the seriousness of liver pathology. Since the possibility of reflecting the occurrence of cell cancer was also assumed (Patent Document 3), first, an assay system using an anti-CSF1R antibody and a WFA lectin was constructed, and using this measurement system, hepatitis patients CSF1R containing WFA-binding sugar chains in body fluid (serum) samples from cancer patients and liver cancer patients, and the amount of CSF1R containing WFA-binding sugar chains varies depending on whether or not hepatocellular carcinoma is present I examined whether or not. As a result, it was found that all hepatitis patients were effective in detecting hepatocellular carcinoma with a significant difference. However, an increase in the amount of CSF1R containing WFA-binding sugar chains is also highly related to the severity of hepatitis and the degree of fibrosis. In severe patients such as cirrhosis patients, there was a deficiency that it was not possible to accurately predict the occurrence of hepatocellular carcinoma.
Therefore, the present inventors focused on a group of patients who suffered from cirrhosis but not hepatocellular carcinoma (LC patients without HCC), followed up these patients, and the subsequent hepatocytes The markers and calculation formulas correlated with the incidence of cancer were examined in detail.
As a result, the ratio of CSF1R containing WFA-binding sugar chains to the total CSF1R in body fluid (serum) (WFA + -CSF1R%), as well as CSF1R containing WFA-binding sugar chains relative to CSF1R containing CSF1R-specific lectin-binding sugar chains The rate was found to be highly correlated with the incidence of hepatocellular carcinoma in patients with cirrhosis. As used herein, “CSF1R-specific lectin” refers to a substance that has no difference in reactivity to sugar chains on CSF1R in body fluids such as serum of healthy persons and patients. Also referred to as “CSF1R-containing sugar chain-specific lectin”, “CSF1R-specific common sugar chain-binding lectin”, “all CSF1R common sugar chain-binding lectin”, and “CSF1R constitutive sugar chain structure-binding lectin”. In other words, it means a lectin that reacts with sugar chains on all CSF1R proteins in body fluids, so instead of measuring the amount of CSF1R containing WFA-binding sugar chains relative to the total amount of CSF1R in body fluids, `` CSF1R-specific lectin-binding sugar chain amount In other words, the ratio of the “WFA-binding sugar chain amount” to “ Since the amount of each lectin-binding sugar chain is proportional to the strength of reactivity to each lectin, the incidence of hepatocellular carcinoma in cirrhosis patients is ultimately reactive to two lectins in the sample fluid This means that it can be measured only by the ratio. The CSF1R-specific lectin typically corresponds to lectins such as RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA shown in Non-Patent Document 1, Fig. 3B. The latter is the ratio of CSF1R containing WFA-binding sugar chains to the total CSF1R in body fluids, just like the former, and together, the “Hepatocellular carcinoma risk assessment index (WFA + -CSF1R%)” Named. Specifically, “WFA-binding sugar chain-containing CSF1R amount / total CSF1R amount × 100” value or “WFA-binding sugar chain-containing CSF1R amount / CSF1R-specific lectin-binding sugar chain-containing CSF1R amount × 100” value, or “WFA-binding sugar chain amount on CSF1R molecule / CSF1R-specific lectin-binding sugar chain amount × 100” value. Using these values and the minimum P-value method in which the incidence of hepatocellular carcinoma in patients with cirrhosis was determined by the log rank test, the optimal cut-off value was 35%. Therefore, when Kaplan-Meier analysis was performed by dividing patients with cirrhosis who did not develop hepatocellular carcinoma into a high value group of 35% or more and a low value group of less than 35%, the 5-year cumulative carcinogenic rate was WFA + -Significantly higher (P = 0.006 or 0.005) was obtained in the high CSF1R% group. This indicates that the value of “WFA + -CSF1R%” is an extremely effective “hepatoma risk assessment index” for cirrhosis patients.
　一方、肝硬変患者の治療後の生存率を検討する中で、WFA結合性糖鎖含有CSF1R量（WFA+-CSF1R値）の数値がそのままで肝硬変患者生存率と高い相関関係にあることを見いだした。具体的には、最小P値法でWFA+-CSF1Rの最適カットオフ値310ng/mlを導き出し、時間依存的ROC曲線で生存率を検討したところ、WFA+-CSF1R値310ng/ml以上でHRが3.63（95％CI1.25-10.54，p=0.011）であり、肝硬変患者の累積生存率をカプラン・マイヤー解析で検討すると、WFA+-CSF1R値高値群は低値群に比較して有意に生存率が低かった。このことは、WFA+-CSF1R値が肝硬変患者の予後を予測する有効な指標となること、すなわち「肝硬変の予後判定指数」となることを示している。 On the other hand, while examining the survival rate after treatment for cirrhosis patients, we found that the value of WFA-binding glycan-containing CSF1R level (WFA + -CSF1R value) is directly correlated with the survival rate of cirrhosis patients. . Specifically, when the optimal cutoff value of 310 ng / ml for WFA + -CSF1R was derived using the minimum P-value method and the survival rate was examined using a time-dependent ROC curve, the HR was higher than the WFA + -CSF1R value of 310 ng / ml. It was 3.63 (95% CI 1.25-10.54, p = 0.011), and the cumulative survival rate of patients with cirrhosis was examined by Kaplan-Meier analysis. The WFA + -CSF1R high-value group was significantly more alive than the low-value group The rate was low. This indicates that the WFA + -CSF1R value is an effective index for predicting the prognosis of cirrhosis patients, that is, the “prognosis index for cirrhosis”.
　さらに、本発明者らは、CSF1R上のWFAレクチン認識糖鎖量及び全CSF1R量の測定値の精度・安定性をあげるため、肝細胞がん発生特異的に増大するCSF1R上のWFAレクチン認識糖鎖構造の解明及び高活性の抗CSF1R検出用抗体の製造を試みた。
　まず、CSF1R遺伝子クローニングして組換えCSF1Rを製造し、CSF1R上の糖鎖結合位置及びそれぞれの糖鎖構造を解明し、CSF1R上のWFAレクチン認識糖鎖構造の特定を行った。
　本発明者らは、以前に組換えWFA遺伝子をクローニングし、C末端側のS-S結合形成を阻止する改変を行って単量体化した組換えWFA（以下、srWFAともいう。）を製造しており、当該srWFAが、LDN糖鎖（非還元末端に「GalNAcβ1-4GlcNAcβ1-R」を有する糖鎖）に特異的に結合することを見いだしていた（特許文献４）。血清試料の抗CSF1R抗体免疫沈降物に当該単鎖組換えWFA（srWFA）を反応させた結果、CSF1R上のWFAレクチン認識糖鎖構造が、srWFAが特異的に認識するLDN糖鎖である可能性が示唆され、LDN欠損株を用いた実験でLDN糖鎖であることがほぼ実証された。
　さらに、組換えCSF1RにGlyco-Ridge法（糖ペプチド・糖鎖構造解析法）を適用して、糖鎖の結合位置を決定した結果、CSF1Rの第１ドメイン（1-87aa）の73位及び第２ドメイン（88-209aa）の153位の少なくとも2箇所に結合していることが解明できた。そして、同時に、市販の組換えCSF1R（Fc融合型（NS0）；R&D Systems社）上の糖鎖構造を調べてみると、市販の組換えCSF1R（NS0）の場合は、いずれのLDN糖鎖も失われていることが解明された。 Furthermore, the present inventors have proposed that the WFA lectin recognition sugars on CSF1R increase specifically in the development of hepatocellular carcinoma in order to increase the accuracy and stability of the measured values of the amount of WFA lectin recognition sugar chains on CSF1R and the total CSF1R amount. Elucidation of chain structure and production of highly active anti-CSF1R detection antibody were attempted.
First, recombinant CSF1R was produced by cloning the CSF1R gene, and the sugar chain binding position and each sugar chain structure on CSF1R were elucidated, and the WFA lectin recognition sugar chain structure on CSF1R was identified.
The present inventors previously produced a recombinant WFA (hereinafter also referred to as srWFA) that has been cloned into a recombinant WFA gene and modified to prevent formation of an SS bond on the C-terminal side. The srWFA was found to specifically bind to LDN sugar chains (sugar chains having “GalNAcβ1-4GlcNAcβ1-R” at the non-reducing end) (Patent Document 4). As a result of reacting the anti-CSF1R antibody immunoprecipitate of the serum sample with the single-chain recombinant WFA (srWFA), the WFA lectin-recognized sugar chain structure on CSF1R may be an LDN sugar chain that srWFA specifically recognizes It was suggested that LDN sugar chains were almost demonstrated in experiments using LDN-deficient strains.
Furthermore, the Glyco-Ridge method (glycopeptide / glycan structure analysis method) was applied to the recombinant CSF1R to determine the binding position of the sugar chain. It was elucidated that it binds to at least two positions at position 153 of two domains (88-209aa). At the same time, when examining the sugar chain structure on the commercially available recombinant CSF1R (Fc fusion type (NS0); R & D Systems), in the case of the commercially available recombinant CSF1R (NS0), any LDN sugar chain It was revealed that it was lost.
　本発明者らの開発したsrWFAレクチンを天然WFAに代えて用いたWFAレクチン－抗CSF1R 抗体ELISAアッセイ系を構築して、天然型WFAレクチンを用いた場合と比較すると感度の良い結果を与えた。また、LDN糖鎖の還元末端側GalNAcを認識するVVAレクチンも、WFAレクチンと同様の結合活性を有することを確認した。つまり、「WFA+-CSF1R量」は、「WFA及び／又はVVA（以下、WFA／VVAと表記することもある。）結合性糖鎖含有CSF1R量」ともいうこともでき、「LDN糖鎖含有CSF1R量」といえる可能性が極めて高い。 A WFA lectin-anti-CSF1R antibody ELISA assay system in which the srWFA lectin developed by the present inventors was used in place of natural WFA was constructed, and a sensitive result was obtained as compared with the case of using the natural WFA lectin. It was also confirmed that the VVA lectin that recognizes GalNAc on the reducing end side of the LDN sugar chain has the same binding activity as the WFA lectin. That is, “WFA + -CSF1R amount” can also be referred to as “WFA and / or VVA (hereinafter sometimes referred to as WFA / VVA) -binding sugar chain-containing CSF1R amount”. There is a very high probability that it can be said to be “CSF1R”.
　次いで、CSF1Rを免疫原として、常法に従い抗CSF1Rモノクローナル抗体を多数作製した。これらの抗CSF1Rモノクローナル抗体のうち、CSF1Rへの親和性の高い33クローンを選択し、直接的ELISAによるCSF1R 結合活性と共に、天然WFAレクチン又は単量体組換えWFA（srWFA）レクチンとのサンドイッチアッセイ系でのCSF1R上のLDN糖鎖の検出性能を検証し、特に検出性能の高い複数の抗CSF1Rモノクローナル抗体をさらに選択した。なお、これら33クローンの抗体産生ハイブリドーマを、それぞれハイブリドーマCSR-1～CSR-33と呼び、各ハイブリドーマが産生するモノクローナル抗体を、それぞれCSR-1～CSR-33抗体と呼ぶ。
　そして、CSF1R上のLDN糖鎖の検出性能の高い抗体の認識ドメインの多くは第２ドメイン又は第３ドメインに集中していることもわかった。また、レクチン－抗体サンドイッチアッセイ系での検出能については、認識ドメインの位置にかかわらず、（表５）中でWFA-CSF1R抗体サンドイッチELISA系で検出可能なものとして示された抗体、具体的には、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、CSR-29抗体が優れており、特にCSR-3、CSR-4、CSR-18、CSR-21、CSR-30抗体の検出能が高かった。なお、典型的な抗CSF1Rモノクローナル抗体産生ハイブリドーマについては、すでにNPMDに寄託している（ハイブリドーマCSR-3、CSR-4、CSR-18、CSR-21及びCSR-30を、それぞれ受領番号：NITE AP-02117～NITE AP-02121として寄託。その後受託番号：NITE P-02117～NITE P-02121が付与され、2016年9月7日付で国際寄託に移管されて、それぞれNITE BP-02117～NITE BP-02121が付与された。）。 Subsequently, a number of anti-CSF1R monoclonal antibodies were prepared according to a conventional method using CSF1R as an immunogen. Among these anti-CSF1R monoclonal antibodies, 33 clones with high affinity for CSF1R were selected, and sandwich assay system with natural WFA lectin or monomeric recombinant WFA (srWFA) lectin together with CSF1R binding activity by direct ELISA The detection performance of LDN sugar chains on CSF1R was verified, and multiple anti-CSF1R monoclonal antibodies with particularly high detection performance were further selected. These 33 clone antibody-producing hybridomas are referred to as hybridomas CSR-1 to CSR-33, respectively, and the monoclonal antibodies produced by each hybridoma are referred to as CSR-1 to CSR-33 antibodies, respectively.
It was also found that many of the antibody recognition domains with high LDN sugar chain detection performance on CSF1R are concentrated in the second domain or the third domain. Regarding the detection ability in the lectin-antibody sandwich assay system, the antibodies shown as detectable in the WFA-CSF1R antibody sandwich ELISA system in (Table 5), regardless of the position of the recognition domain, Are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13, The CSR-26, CSR-27, and CSR-29 antibodies were superior, especially the ability to detect CSR-3, CSR-4, CSR-18, CSR-21, and CSR-30 antibodies. Note that typical anti-CSF1R monoclonal antibody-producing hybridomas have already been deposited with NPMD (hybridomas CSR-3, CSR-4, CSR-18, CSR-21 and CSR-30, respectively, receipt number: NITE AP Deposited as -02117-NITE AP-02121, and then given the accession numbers: NITE P-02117-NITE P-02121, transferred to the international deposit on September 7, 2016, respectively, NITE BP-02117-NITE BP- 02121 was granted.).
　これらの抗CSF1Rモノクローナル抗体を、CSF1R分子検出用アッセイ系に、及び／又は天然WFAレクチン、リコンビナントWFA、単量体リコンビナントレクチン（srWFA）もしくはVVAレクチンと共にサンドイッチELISAアッセイ系に用いることで、被験者の体液（血清）中のCSF1R量並びにWFA及び／又はVVA（WFA／VVA）結合性糖鎖含有CSF1R量をより正確に測定できることが示唆される。実際に、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30 、CSR-5及びCSR-6抗体では肝硬変を背景とする肝細胞がん患者の体液（血清）試料でも健常人と比較してCSF1Rシグナルの増強が検出できることを確認している。すなわち、WFA／VVA結合性糖鎖結合性レクチンと共にCSR-3もしくはCSR-4、CSR-18、CSR-21、CSR-30抗体などと組み合わせたサンドイッチELISAアッセイ系を用いることで、正確なWFA+-CSF1Rの数値が測定できた。このことは、本発明の肝硬変患者の肝細胞がん発生のリスク予測及び予後の予測が精度良く行うことができるだけでなく、広く肝疾患患者又はその疑いのある患者に対し、肝疾患の有無又は重篤度（線維化度）の判定用に用いることもできることを意味する。
　以上の知見が得られたことで、本願発明が完成した。 By using these anti-CSF1R monoclonal antibodies in an assay system for detecting CSF1R molecules and / or in sandwich ELISA assay systems together with natural WFA lectin, recombinant WFA, monomeric recombinant lectin (srWFA) or VVA lectin, It is suggested that the amount of CSF1R in (serum) and the amount of CSF1R containing WFA and / or VVA (WFA / VVA) -binding sugar chain can be measured more accurately. In fact, CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5 and CSR-6 antibodies are healthy even in body fluid (serum) samples of patients with hepatocellular carcinoma against cirrhosis. It has been confirmed that an increase in CSF1R signal can be detected compared to humans. In other words, by using a sandwich ELISA assay system combined with CSR-3 or CSR-4, CSR-18, CSR-21, CSR-30 antibody together with WFA / VVA-binding sugar chain-binding lectin, accurate WFA + -The value of CSF1R could be measured. This can not only accurately predict the risk of hepatocellular carcinoma occurrence and prognosis of the hepatic cirrhosis patient of the present invention, but also widely present or suspected of having liver disease or not. It means that it can also be used for determination of severity (fibrosis degree).
By obtaining the above knowledge, the present invention was completed.
　すなわち、本願発明は、以下の通りである。
〔１〕　肝硬変患者における肝細胞がんの発症リスク値の算出方法であって、（１）～（４）の工程を含む算出方法；
（１）肝硬変患者の被験者から採取された一定容量の体液試料（以下、単に被検試料ともいう。）中の全CSF1R量（Ａ）を測定する工程、
（２）被検試料中のWFA／VVA結合性糖鎖含有CSF1R量（Ｂ）を測定する工程、及び
（３）全CSF1Rに占めるWFA／VVA結合性糖鎖を含有するCSF1R量の比率（Ｃ）を、「Ｃ（％）＝（Ｂ）／（Ａ）×100」として算出する工程、
（４）工程（３）で得られたＣ％の値を被験者の肝細胞がんの発症リスク値と決定する工程。
　ここで、肝細胞がん発がんリスク指数値（Ｃ％）は（WFA+-CSF1R％）と表記されることもある。
〔２〕　（１）の全CSF1R量を測定する工程が、少なくとも2種類の抗CSF1R抗体を用いるサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、精製CSF1R量を測定することを特徴とする、前記〔１〕に記載の方法。
〔３〕　（１）の被検試料中の全CSF1R量（Ａ）を測定する工程が、被検試料中のCSF1R特異的レクチン結合性糖鎖含有CSF1R量を測定するものであり、少なくともCSF1R特異的レクチンと、抗CSF1R抗体とを含むサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、CSF1R特異的レクチンと結合する精製CSF1R量を測定することを特徴とする、前記〔１〕に記載の方法。
〔４〕　CSF1R特異的レクチンが、RCA120, DSA, PHA-E4、SNA、SSA、TJA-I、LEL、STL及びConAからなる群から選択される少なくとも１つのレクチンである、前記〔３〕に記載の方法。
〔５〕　（２）のWFA／VVA結合性糖鎖含有CSF1R量（Ｂ）を測定する工程が、少なくともWFA／VVA結合性糖鎖特異的レクチンと、抗CSF1R抗体とを含むサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、WFA／VVAレクチンと結合する精製CSF1R量を測定することを特徴とする、前記〔１〕～〔４〕のいずれか一項に記載の方法。
〔６〕　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAのいずれかのレクチンから選択される少なくとも１つのレクチンである、前記〔５〕に記載の方法。
〔７〕　工程（１）及び工程（２）が、少なくともCSF1R特異的レクチン及びWFA／VVAレクチンと抗CSF1R抗体とを用いて同時に行われる工程であって、両レクチン及び抗CSF1R抗体とを含む同一のサンドイッチアッセイ系を用いて測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製した後、各レクチンと結合するCSF1R量を同一のアッセイ系で測定することを特徴とする、前記〔３〕～〔６〕のいずれかに記載の方法。
〔８〕　肝硬変患者における肝細胞がんの発症リスク値を判定する方法であって、（１）～（３）の工程を含む方法；
（１）肝硬変患者である被験者の肝細胞がんの発症リスク値（Ｃ％）を前記〔１〕～〔７〕のいずれかに記載の方法に従い算出する工程、
（２）あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者から採取された各体液試料に対し、工程（１）と同じ算出工程により、各々の全CSF1Rに占めるWFA／VVA結合性糖鎖を含有するCSF1R量の比率（Ｃｎ）と、各々の患者をフォローアップして得た肝細胞がん発症率データと対比させて肝細胞がん発症の最適カットオフ値（Ｍ％）を算出する工程、
（３）工程（１）で算出された肝細胞がんの発症リスク値（Ｃ％）を（２）で算出された最適カットオフ値（Ｍ％）と比較して上回っている場合に被験者の肝細胞がん発症リスクが有意に高いと判定し、最適カットオフ値未満であれば発症リスクが有意に低いと判定する方法。
　ここで、最適カットオフ値は、あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者をフォローアップして得た発がん率データに基づいて算出された値であり、例えば、発がん率データに対し、ログランク検定で決定した最小P値法を適用し、上下10%を除外することで求めることができる。なお、十分な母数とは、10～6000例、10～5000例、10～4000例、10～3000例、10～1000例、好ましくは30～3000例、30～2000例、30～1000例、より好ましくは40～2000例、40～1000例、50～2000例、50～1000例、さらに好ましくは50～500例、100～500例を指す。
　また、本判定方法は、肝硬変患者における肝細胞がんの発症リスクを予測するための肝細胞がん発症リスク指数（Ｃ％）の測定方法、又は肝細胞がんの発症リスクを診断するための資料（情報）を提供する方法などと表現することもできる。
〔９〕　前記最適カットオフ値が35.0±10.0％の値である前記〔８〕に記載の方法。
〔１０〕　肝硬変患者における予後判定指数値の算出方法であって、（１）及び（２）の工程を含む方法；
（１）肝硬変患者である被験者から採取された一定容量の体液試料（被検試料）中のWFA／VVA結合性糖鎖を含有するCSF1R量（Ｂ）を測定する工程、
（２）工程（１）で得られたＢng/mlの値を、被験者の予後判定指数値と決定する工程。
　ここで、予後判定指数値（Ｂng/ml）は、（WFA+-CSF1R ng/ml）と表記されることもある。
〔１１〕　（１）のWFA／VVA結合性糖鎖含有CSF1R量（Ｂ）を測定する工程が、少なくともWFA／VVAレクチンと、抗CSF1R抗体とを含むサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、WFA／VVAレクチンと結合する精製CSF1R量を測定することを特徴とする、前記〔１０〕に記載の方法。
〔１２〕　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAのいずれかのレクチンから選択される少なくとも１つのレクチンである、前記〔１１〕に記載の方法。
〔１３〕　肝硬変患者における予後を判定する方法であって、（１）～（３）の工程を含む方法；
（１）肝硬変患者である被験者の予後判定指数値（Ｂng/ml）を前記〔１０〕～〔１２〕のいずれかに記載の方法に従い算出する工程、
（２）あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者から採取された各体液試料に対し、工程（１）と同じ算出工程により、各々のWFA／VVA結合性糖鎖を含有するCSF1R量（Ｂｎ）と、各々の患者をフォローアップして得た累積生存率データと対比させて肝細胞がん患者予後の最適カットオフ値（Ｎng/ml）を算出する工程、
（３）工程（１）で算出された予後判定指数値（Ｂng/ml）を（２）で算出された最適カットオフ値（Ｎng/ml）と比較して上回っている場合に被験者の被験者の予後が有意に悪いと判定し、最適カットオフ値未満であれば被験者の予後が有意に良いと判定する方法。
　ここで、最適カットオフ値は、あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者をフォローアップして得た累積生存率に基づいて算出された値であって、例えば、５年累積生存率データに対し、ログランク検定で決定した最小P値法を適用し、上下10%を除外することで求めることができる。なお、十分な母数とは、10～6000例、10～5000例、10～4000例、10～3000例、10～1000例、好ましくは30～3000例、30～2000例、30～1000例、より好ましくは40～2000例、40～1000例、50～2000例、50～1000例、さらに好ましくは50～500例、100～500例を指す。また、予後判定指数値（Ｂng/ml、WFA+-CSF1R ng/ml）の値は、COIとして算出して判定に利用することも出来る。
　また、本判定方法は、肝硬変患者の予後を予測するための予後判定指数（Ｂng/ml）の測定方法、又は肝硬変患者の予後を診断するための資料を提供する方法と表現することもできる。
〔１４〕　前記最適カットオフ値が310±100ng/mlの値である前記〔１３〕に記載の方法。
〔１５〕　WFA／VVA結合性糖鎖含有CSF1Rを検出又は定量するためのレクチン－抗体サンドイッチアッセイであって、
WFA／VVAレクチンと、
CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体とを用い、（１）～（３）の工程を含むアッセイ；
（１）液相中で前記レクチン又は前記抗CSF1R抗体のいずれかと被検試料を接触させ、被検試料中のCSF1Rとの複合体を形成させる工程、
（２）（１）で得られたレクチン又は抗体とのCSF1R複合体を、分離し、又は分離せずに他方が溶解もしくは分散されている検出用液相中で、他方とCSF1R複合体を結合させ、レクチン及び抗体でサンドイッチされたCSF1R複合体を得る工程、
（３）（２）で得られたレクチン及び抗体サンドイッチCSF1R複合体量を検出又は定量する工程。
　ここで、典型的なサンドイッチアッセイは、固相-液相サンドイッチアッセイ、特にサンドイッチELISAであるが、サンドイッチELISAには限られないことはもちろん、固相-液相ではなく液相-液相であってもよい。固相-液相サンドイッチアッセイの場合は、レクチン又は抗体のいずれかが捕捉側に設けられ、他方が検出用液相側に溶解もしくは分散されている。また、サンドイッチELISAの場合は、「捕捉側」は「固相面」となる。
〔１６〕　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンである、前記〔１５〕に記載のアッセイ。
〔１７〕　WFA／VVA結合性糖鎖含有CSF1Rを検出又は定量するためのレクチン－抗体サンドイッチアッセイ用キットであって、（１）及び（２）を含む、キット；
（１）WFA／VVAレクチン、
（２）CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体。
〔１８〕　さらに、以下の（３）を含む、前記〔１７〕に記載のキット；
（３）WFA／VVA結合性糖鎖含有CSF1R及び／又はWFA／VVA結合性糖鎖非含有CSF1Rからなる標準物質。
〔１９〕　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンである、前記〔１８〕又は〔１９〕に記載のキット。
〔２０〕　レクチン－抗体サンドイッチアッセイが、被験者由来の体液試料に適用し、体液試料中のWFA／VVA結合性糖鎖含有CSF1Rを検出又は定量するためのアッセイである、前記〔１７〕～〔１９〕のいずれかに記載のキット。
〔２１〕　CSR-3（国際受託番号：NITE BP-02117）、CSR-4（国際受託番号：NITE BP-02118）CSR-18（国際受託番号：NITE BP-02119）、CSR-21（国際受託番号：NITE BP-02120）、CSR-30（国際受託番号：NITE BP-02121）からなる群から選択されるいずれか１つのハイブリドーマから産生される抗CSF1R抗体又はその抗体結合性フラグメント。
〔２２〕　肝硬変患者である被験者の肝細胞がん発症リスク値及び／又は予後判定値を算出するためのキットであって、（１）及び（２）のレクチンを含むことを特徴とするキット；
（１）WFA／VVAレクチン、
（２）CSF1R特異的レクチン。
　なお、当該キットには、さらにWFA／VVA結合性糖鎖含有CSF1R及び／又はWFA／VVA結合性糖鎖非含有CSF1Rからなる標準物質を含んでもよい。
〔２３〕　さらに、（３）を含むことを特徴とする前記〔２２〕に記載のキット；
（３）抗CSF1R抗体又はその抗体結合性フラグメント。
〔２４〕　（１）のWFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンであり、
（２）のCSF1R特異的レクチンが、RCA120, DSA, PHA-E4、SNA、SSA、TJA-I、LEL、STL及びConAからなる群から選択される少なくとも１つのレクチンである、前記〔２２〕又は〔２３〕に記載のキット。
〔２５〕　（１）又は（２）のレクチンのいずれか一方がWFA／VVA結合性糖鎖含有CSF1Rの捕捉用に設けられた固相に結合されており、かつ他方が検出用の液相に溶解又は分散されている、前記〔２２〕～〔２４〕のいずれかに記載のキット。
〔２６〕　（１）及び（２）のレクチンの両方が同一もしくは異なるレクチンアレイ上に結合している、前記〔２２〕～〔２４〕のいずれかに記載のキット。
〔２７〕　肝硬変患者である被験者の肝細胞がん発がんリスク及び／又は予後を判定するためのキットであって、（１）～（３）を含むことを特徴とする判定用キット；
（１）WFA／VVAレクチン、
（２）CSF1R特異的レクチン。
（３）抗CSF1R抗体又はその抗体結合性フラグメント。
　なお、当該キットには、さらにWFA／VVA結合性糖鎖含有CSF1R及び／又はWFA／VVA結合性糖鎖非含有CSF1Rからなる標準物質を含んでもよい。
〔２８〕　（１）のWFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンであり、
（２）のCSF1R特異的レクチンが、RCA120, DSA, PHA-E4、SNA、SSA、TJA-I、LEL、STL及びConAからなる群から選択される少なくとも１つのレクチンであり，及び
（３）の抗CSF1R抗体が、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体である、請求項２７に記載の判定用キット。
〔２９〕　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するための方法であって、（１）及び（２）の工程を含むことを特徴とする方法；
（１）被験者由来の体液試料から抗CSF1R抗体を用いてCSF1Rタンパク質を分離精製する工程、
（２）（１）で分離精製されたCSF1R上のWFA／VVA結合性糖鎖含有量及びCSF1R特異的糖鎖含有量を、測定する工程。
〔３０〕　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するための方法であって、（１）及び（２）の工程を含むことを特徴とする方法；
（１）被験者由来の体液試料から天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンを用いてWFA／VVA結合性糖鎖を含有する糖タンパク質を分離する工程、
（２）（１）で分離精製されたWFA／VVA結合性糖鎖を含有する糖タンパク質から抗CSF1R抗体を用いてCSF1Rタンパク質を検出又は定量する工程、
ここで、抗CSF1R抗体は、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体である。
〔３１〕　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するための方法であって、被験者由来の体液試料に対し、（１）及び（２）を用いたレクチン－抗体サンドイッチアッセイを行う工程を含むことを特徴とする方法；
（１）WFA／VVAレクチン、
（２）CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体。
〔３２〕　肝細胞がん未罹患肝硬変患者における肝細胞がん発症リスクを判定するための方法であって、（１）及び（２）の工程を含むことを特徴とする方法；
（１）肝細胞がん未罹患肝硬変患者である被験者から採取された体液試料中のWFA／VVA結合性糖鎖を含有するCSF1R量、及び全CSF1R量を測定する工程、
（２）（１）で得られた測定値をもとに、全CSF1Rに占めるWFA／VVA結合性糖鎖を有するCSF1R量の比率を算出する工程、
（３）あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者から採取された体液試料に対し、前記（１）及び（２）と同じ工程により、全CSF1Rに占めるWFA／VVA結合性糖鎖を有するCSF1R量の比率を全患者についてそれぞれ算出する工程、
（４）（３）で用いた全患者をフォローアップして得た発がん率データに基づいて最適カットオフ値を算出する工程、
（５）（２）で算出された被験者の比率の値を（４）で算出された最適カットオフ値と比較して上回っている場合に肝細胞がん発症リスクが高いと判定する方法。 That is, the present invention is as follows.
[1] A method for calculating a risk of developing hepatocellular carcinoma in a cirrhotic patient, comprising the steps of (1) to (4);
(1) a step of measuring the total amount of CSF1R (A) in a constant volume of body fluid sample (hereinafter also simply referred to as a test sample) collected from a subject of a cirrhotic patient;
(2) a step of measuring the amount of CSF1R containing WFA / VVA-binding sugar chains (B) in a test sample, and (3) the ratio of the amount of CSF1R containing WFA / VVA-binding sugar chains to the total CSF1R (C ) As “C (%) = (B) / (A) × 100”,
(4) A step of determining the value of C% obtained in step (3) as the risk of developing hepatocellular carcinoma of the subject.
Here, the hepatocellular carcinoma carcinogenic risk index value (C%) may be expressed as (WFA + -CSF1R%).
[2] The step of measuring the total CSF1R amount in (1) is measured by a sandwich assay system using at least two types of anti-CSF1R antibodies, or purified from a test sample using anti-CSF1R antibodies and purified. The method according to [1] above, wherein the amount of CSF1R is measured.
[3] The step (1) of measuring the total CSF1R amount (A) in the test sample is a method for measuring the CSF1R-specific lectin-binding sugar chain-containing CSF1R amount in the test sample. CSF1R is purified from a test sample using an anti-CSF1R antibody, and the amount of purified CSF1R binding to a CSF1R-specific lectin is measured. The method according to [1] above.
[4] The CSF1R-specific lectin is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA. the method of.
[5] The step of measuring the amount (B) of WFA / VVA-binding sugar chain-containing CSF1R in (2) is measured by a sandwich assay system containing at least a WFA / VVA-binding sugar chain-specific lectin and an anti-CSF1R antibody. Or purifying CSF1R from a test sample using an anti-CSF1R antibody, and measuring the amount of purified CSF1R that binds to WFA / VVA lectin, any one of [1] to [4] The method according to item.
[6] The method according to [5] above, wherein the WFA / VVA lectin is at least one lectin selected from any one of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
[7] Steps (1) and (2) are performed simultaneously using at least a CSF1R-specific lectin and a WFA / VVA lectin and an anti-CSF1R antibody, and include both the lectin and the anti-CSF1R antibody. The CSF1R bound to each lectin is measured with the same assay system after the CSF1R is purified using an anti-CSF1R antibody from a test sample, or measured using the same assay system. [3] The method according to any one of [6].
[8] A method for determining the risk of developing hepatocellular carcinoma in a cirrhotic patient, comprising the steps of (1) to (3);
(1) a step of calculating a risk of developing hepatocellular carcinoma (C%) of a subject who has cirrhosis according to the method according to any one of [1] to [7],
(2) A WFA / VVA-binding glycan occupying in each CSF1R is calculated for each body fluid sample collected from a liver parameter cirrhosis patient who does not have hepatocellular carcinoma in advance by the same calculation step as in step (1). The optimal cut-off value (M%) for the development of hepatocellular carcinoma is calculated by comparing the ratio (Cn) of the amount of CSF1R containing HC1 and the hepatocellular carcinoma incidence data obtained by following up each patient Process,
(3) If the risk of developing hepatocellular carcinoma (C%) calculated in step (1) is higher than the optimal cutoff value (M%) calculated in (2), A method for determining that the risk of developing hepatocellular carcinoma is significantly high, and determining that the risk of developing is significantly low if the risk is less than the optimum cutoff value.
Here, the optimal cut-off value is a value calculated based on the carcinogenic rate data obtained by following up a liver parameter cirrhosis patient who does not have a sufficient population of hepatocellular carcinoma in advance. It can be obtained by applying the minimum P-value method determined by the log rank test and excluding the upper and lower 10%. The sufficient number of parameters is 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 1000, preferably 30 to 3000, 30 to 2000, 30 to 1000. More preferably, it refers to 40 to 2000, 40 to 1000, 50 to 2000, 50 to 1000, more preferably 50 to 500, and 100 to 500.
In addition, this determination method is a method for measuring the risk of developing hepatocellular carcinoma (C%) for predicting the risk of developing hepatocellular carcinoma in cirrhosis patients, or for diagnosing the risk of developing hepatocellular carcinoma. It can also be expressed as a method of providing material (information).
[9] The method according to [8], wherein the optimum cut-off value is 35.0 ± 10.0%.
[10] A method for calculating a prognostic index value in a patient with cirrhosis, comprising the steps of (1) and (2);
(1) a step of measuring the amount of CSF1R (B) containing a WFA / VVA-binding sugar chain in a constant volume of body fluid sample (test sample) collected from a subject who has cirrhosis;
(2) A step of determining the value of Bng / ml obtained in step (1) as the prognosis determination index value of the subject.
Here, the prognosis determination index value (Bng / ml) may be expressed as (WFA + -CSF1R ng / ml).
[11] The step of (1) measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain (B) is measured by a sandwich assay system containing at least a WFA / VVA lectin and an anti-CSF1R antibody, or a test The method according to [10] above, wherein CSF1R is purified from a sample using an anti-CSF1R antibody, and the amount of purified CSF1R that binds to WFA / VVA lectin is measured.
[12] The method according to [11] above, wherein the WFA / VVA lectin is at least one lectin selected from any one of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
[13] A method for determining the prognosis in a cirrhotic patient, comprising the steps of (1) to (3);
(1) calculating a prognostic index value (Bng / ml) of a subject who is a cirrhotic patient according to the method according to any one of [10] to [12],
(2) CSF1R containing each WFA / VVA-binding glycan is obtained by the same calculation process as in step (1) on each body fluid sample collected from a liver cirrhosis patient who does not have hepatocellular carcinoma in advance. Calculating the optimal cutoff value (Nng / ml) of hepatocellular carcinoma patient prognosis by comparing the amount (Bn) and cumulative survival data obtained by following up each patient;
(3) When the prognosis determination index value (Bng / ml) calculated in step (1) is higher than the optimal cutoff value (Nng / ml) calculated in (2), A method of determining that the prognosis is significantly poor and that the prognosis of the subject is significantly good if it is less than the optimum cutoff value.
Here, the optimal cut-off value is a value calculated based on a cumulative survival rate obtained by following up a liver parameter cirrhosis patient who does not have a sufficient number of hepatocellular carcinomas in advance. The rate data can be obtained by applying the minimum P-value method determined by the log rank test and excluding the upper and lower 10%. The sufficient number of parameters is 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 1000, preferably 30 to 3000, 30 to 2000, 30 to 1000. More preferably, it refers to 40 to 2000, 40 to 1000, 50 to 2000, 50 to 1000, more preferably 50 to 500, and 100 to 500. Further, the prognosis determination index value (Bng / ml, WFA + -CSF1R ng / ml) can be calculated as COI and used for the determination.
This determination method can also be expressed as a method for measuring a prognosis determination index (Bng / ml) for predicting the prognosis of a cirrhosis patient or a method for providing data for diagnosing the prognosis of a cirrhosis patient.
[14] The method according to [13], wherein the optimum cutoff value is 310 ± 100 ng / ml.
[15] A lectin-antibody sandwich assay for detecting or quantifying CSF1R containing a WFA / VVA-binding sugar chain,
WFA / VVA lectin,
CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13, CSR- An assay comprising the steps of (1) to (3) using at least one anti-CSF1R antibody selected from the group consisting of 26, CSR-27, and CSR-29 antibodies;
(1) contacting the test sample with either the lectin or the anti-CSF1R antibody in a liquid phase to form a complex with CSF1R in the test sample;
(2) The CSF1R complex with the lectin or antibody obtained in (1) is separated, or the other is bound to the CSF1R complex in the detection liquid phase in which the other is dissolved or dispersed. Obtaining a CSF1R complex sandwiched with a lectin and an antibody,
(3) A step of detecting or quantifying the amount of the lectin and antibody sandwich CSF1R complex obtained in (2).
Here, a typical sandwich assay is a solid phase-liquid phase sandwich assay, particularly a sandwich ELISA, but is not limited to a sandwich ELISA, and of course is a liquid phase-liquid phase rather than a solid phase-liquid phase. May be. In the case of a solid-liquid phase sandwich assay, either a lectin or an antibody is provided on the capture side, and the other is dissolved or dispersed on the detection liquid phase side. In the case of sandwich ELISA, the “capture side” is the “solid phase surface”.
[16] The assay according to [15], wherein the WFA / VVA lectin is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
[17] A lectin-antibody sandwich assay kit for detecting or quantifying CSF1R containing a WFA / VVA-binding sugar chain, comprising (1) and (2);
(1) WFA / VVA lectin,
(2) CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13 At least one anti-CSF1R antibody selected from the group consisting of antibodies, CSR-26, CSR-27, and CSR-29.
[18] The kit according to [17], further comprising the following (3):
(3) A standard substance comprising a CSF1R containing a WFA / VVA-binding sugar chain and / or a CSF1R containing no WFA / VVA-binding sugar chain.
[19] The kit according to [18] or [19], wherein the WFA / VVA lectin is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
[20] The above-mentioned [17] to [19], wherein the lectin-antibody sandwich assay is an assay for detecting or quantifying WFA / VVA-binding sugar chain-containing CSF1R in a body fluid sample applied to a body fluid sample derived from a subject. ] The kit in any one of.
[21] CSR-3 (International accession number: NITE BP-02117), CSR-4 (International accession number: NITE BP-02118) CSR-18 (International accession number: NITE BP-02119), CSR-21 (International accession number) Number: NITE BP-02120), CSR-30 (International Accession Number: NITE BP-02121), an anti-CSF1R antibody produced from any one of the hybridomas or an antibody-binding fragment thereof.
[22] A kit for calculating a risk of developing hepatocellular carcinoma and / or a prognostic value of a subject who is a cirrhotic patient, the kit comprising the lectin of (1) and (2);
(1) WFA / VVA lectin,
(2) CSF1R specific lectin.
The kit may further contain a standard substance comprising CSF1R containing WFA / VVA-binding sugar chain and / or CSF1R containing no WFA / VVA-binding sugar chain.
[23] The kit according to [22], further comprising (3);
(3) An anti-CSF1R antibody or an antibody-binding fragment thereof.
[24] The WFA / VVA lectin of (1) is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA,
The CSF1R-specific lectin of (2) is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA [22] or [23] The kit according to [23].
[25] Either one of the lectins of (1) or (2) is bound to a solid phase provided for capturing WFA / VVA-binding sugar chain-containing CSF1R, and the other is in a liquid phase for detection. The kit according to any one of [22] to [24], wherein the kit is dissolved or dispersed.
[26] The kit according to any one of [22] to [24] above, wherein both the lectins of (1) and (2) are bound on the same or different lectin arrays.
[27] A kit for determining the risk of hepatocellular carcinoma carcinogenesis and / or prognosis of a subject who has cirrhosis, comprising (1) to (3);
(1) WFA / VVA lectin,
(2) CSF1R specific lectin.
(3) An anti-CSF1R antibody or an antibody-binding fragment thereof.
The kit may further contain a standard substance comprising CSF1R containing WFA / VVA-binding sugar chain and / or CSF1R containing no WFA / VVA-binding sugar chain.
[28] The WFA / VVA lectin of (1) is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA,
(2) the CSF1R-specific lectin is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA, and (3) Anti-CSF1R antibodies are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR The determination kit according to claim 27, wherein the determination kit is at least one anti-CSF1R antibody selected from the group consisting of -13, CSR-26, CSR-27, and CSR-29 antibodies.
[29] A method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a cirrhotic patient, comprising the steps of (1) and (2);
(1) a step of separating and purifying CSF1R protein from a body fluid sample derived from a subject using an anti-CSF1R antibody,
(2) A step of measuring the WFA / VVA-binding sugar chain content and the CSF1R-specific sugar chain content on the CSF1R separated and purified in (1).
[30] A method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a cirrhotic patient, comprising the steps of (1) and (2);
(1) A glycoprotein containing a WFA / VVA-binding sugar chain using at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA from a body fluid sample derived from a subject. Separating,
(2) a step of detecting or quantifying the CSF1R protein using the anti-CSF1R antibody from the glycoprotein containing the WFA / VVA-binding sugar chain separated and purified in (1),
Here, anti-CSF1R antibodies are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR- 9, at least one anti-CSF1R antibody selected from the group consisting of CSR-13, CSR-26, CSR-27, and CSR-29 antibodies.
[31] A method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a patient with cirrhosis, wherein the lectin-antibody uses (1) and (2) for a body fluid sample derived from the subject Performing a sandwich assay;
(1) WFA / VVA lectin,
(2) CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13 At least one anti-CSF1R antibody selected from the group consisting of antibodies, CSR-26, CSR-27, and CSR-29.
[32] A method for determining the risk of developing hepatocellular carcinoma in a cirrhosis patient who does not have hepatocellular carcinoma, comprising the steps of (1) and (2);
(1) a step of measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain and the total amount of CSF1R in a body fluid sample collected from a subject who is a liver cirrhosis patient who does not have hepatocellular carcinoma;
(2) calculating the ratio of the amount of CSF1R having a WFA / VVA-binding sugar chain to the total CSF1R based on the measurement value obtained in (1),
(3) A WFA / VVA-binding glycan occupying in the total CSF1R is obtained from a bodily fluid sample collected from a cirrhosis patient who does not have hepatocellular carcinoma in advance by the same process as (1) and (2) above. Calculating the ratio of CSF1R amount for all patients,
(4) calculating an optimal cutoff value based on carcinogenicity data obtained by following up all patients used in (3);
(5) A method for determining that the risk of developing hepatocellular carcinoma is high when the value of the ratio of subjects calculated in (2) is higher than the optimum cutoff value calculated in (4).
　本発明においては、肝硬変患者の肝細胞がん発生リスクと極めて相関性の高い「肝細胞がんリスク判定指数（WFA+-CSF1R％）」を提供できたことで、当該判定指数に応じた発がんリスク割合を示すことができた。すなわち、血液試料による肝硬変患者の肝細胞がん発生のリスク判定方法を提供できた。当該方法を用いることで、肝硬変患者など重篤な肝疾患患者に対しても、簡便な血液検査によってほぼ正確に発がんリスクが把握できるから、負担の大きな侵襲性の煩雑な検査が不要又は回数を減らすことが可能となった。 In the present invention, the “hepatocellular carcinoma risk determination index (WFA + -CSF1R%)” that is highly correlated with the risk of developing hepatocellular carcinoma in cirrhosis patients can be provided, and carcinogenesis according to the determination index The risk ratio could be shown. That is, a risk determination method for the occurrence of hepatocellular carcinoma in cirrhosis patients using blood samples could be provided. By using this method, even for patients with severe liver disease such as cirrhosis patients, the risk of cancer can be grasped almost accurately with a simple blood test. It became possible to reduce.
　また、本発明は、WFA結合性糖鎖含有CSF1R量（WFA+-CSF1R値）が、肝硬変の予後と極めて相関の高い「肝硬変の予後判定指数」となることを初めて見いだしたものであり、当該数値範囲に応じた生存率を示すことができた。すなわち、血液試料による肝硬変患者の予後の正確な判定方法を提供することができた。 In addition, the present invention has been found for the first time that the amount of CSF1R containing WFA-binding sugar chains (WFA + -CSF1R value) is a "prognosis index for cirrhosis" that is highly correlated with the prognosis of cirrhosis. The survival rate according to the numerical range could be shown. That is, it was possible to provide an accurate method for determining the prognosis of a cirrhotic patient using a blood sample.
　同時に、本発明のCSF1R上のWFA結合性糖鎖検出用レクチンとして、単量体リコンビナントWFAレクチン試薬と共に、VVAレクチン試薬が有効であることを見いだした。あわせてWFA及び／又はVVA（WFA／VVA）結合性糖鎖を含有するCSF1R検出用の抗体として高い結合活性を有する複数の抗CSF1Rモノクローナル抗体を提供できた。これら抗体をキットとして組み合わせることで、より正確な「肝細胞がん発症リスク判定指数」または「肝硬変の予後判定指数」を算出することが可能となり、肝硬変患者における肝細胞がん発症リスク判定の精度及び肝硬変の予後判定の精度を高めることができる。 At the same time, the present inventors have found that a VVA lectin reagent is effective together with a monomeric recombinant WFA lectin reagent as a lectin for detecting a WFA-binding sugar chain on CSF1R of the present invention. In addition, a plurality of anti-CSF1R monoclonal antibodies having high binding activity could be provided as antibodies for detecting CSF1R containing WFA and / or VVA (WFA / VVA) -binding sugar chains. Combining these antibodies as a kit makes it possible to calculate a more accurate “hepatoma risk assessment index” or “cirrhosis prognostic index”, and the accuracy of hepatocellular carcinoma risk assessment in patients with cirrhosis And the accuracy of the prognosis determination of cirrhosis can be improved.
免疫組織化学染色解析：肝細胞がん組織でCSF1RおよびWFAエピトープの発現Immunohistochemical staining analysis: CSF1R and WFA epitope expression in hepatocellular carcinoma tissue 免疫組織化学染色解析：HCC組織アレイを用いた組織化学染色解析（抗CSF1R抗体とWFA)Immunohistochemical staining analysis: Histochemical staining analysis using HCC tissue array (anti-CSF1R antibody and WFA) 分析プロセスフローチャート　本コホートに参加した214名のHCV由来肝炎患者の内訳を示す。本実施例では、肝細胞がんを発症していない肝硬変患者（非HCC-LC患者、56名）を選択し、さらにバリデーションコホートとして別の無作為に選ばれた45名の非HCC-LC患者群をあわせて評価した。Analysis process flowchart Shows the breakdown of 214 HCV-derived hepatitis patients who participated in this cohort. In this example, cirrhosis patients who did not develop hepatocellular carcinoma (non-HCC-LC patients, 56 patients) were selected, and 45 other non-HCC-LC patients randomly selected as a validation cohort Groups were evaluated together. ログランク検定で求められた最小P値法による累積発がん率予測のためのWFA+-CSF1R％の最適カットオフ値。The optimal cutoff value of WFA + -CSF1R% for predicting the cumulative cancer rate by the minimum P-value method determined by the log rank test. （a）WFA+-CSF1R値とWFA+-CSF1R(％)値との相関の二次元scatter plot（散布図）を、101名の肝細胞がん未罹患LC患者由来血清試料に対して実施した。WFA+-CSF1R値とWFA+-CSF1R(％)値との相関は、回帰曲線でY=7.9663X+18.735、R2=0.6488で表される。好ましい相関を示しているものの、大きく逸脱するケースがいくつか見られた。（b）WFA+-CSF1R(％)値と発がん日数についての相関；41名の肝細胞がん未罹患LC患者ログランクに関し、WFA+-CSF1R(％)値と発がんまでの日数の相関は、回帰曲線でY＝-0.8618X+59.681、R2=0.1448で表される。The (a) WFA + -CSF1R value and WFA + -CSF1R (%) two-dimensional scatter plot (Scatter) correlation with values, was performed on hepatocellular carcinoma unaffected LC patients from serum samples 101 people . The correlation between the WFA + -CSF1R value and the WFA + -CSF1R (%) value is represented by Y = 7.9663X + 18.735, R 2 = 0.6488 in the regression curve. Although it showed a favorable correlation, there were some cases that deviated significantly. (B) Correlation between WFA + -CSF1R (%) value and the number of days of carcinogenesis; With regard to 41 hepatocellular carcinoma non-affected LC patients log rank, the correlation between WFA + -CSF1R (%) value and the number of days until carcinogenesis is The regression curve is represented by Y = −0.8618X + 59.681, R 2 = 0.1448. 肝細胞がん未罹患の慢性肝炎患者におけるWFA+-CSF1R(％)値は、F1～F3の肝線維化レベルにかかわらず統計学的に有意な差はなく、肝硬変患者でのWFA+-CSF1R(％)値は慢性肝炎患者と有意な差があった。さらに、肝細胞がんを発症しなかった肝硬変患者と発症した肝硬変患者のWFA+-CSF1R(％)値にも有意な差があった。WFA + -CSF1R (%) in chronic hepatitis patients without hepatocellular carcinoma is not statistically significant regardless of liver fibrosis levels from F1 to F3, and WFA + -CSF1R in patients with cirrhosis (%) Value was significantly different from chronic hepatitis patients. Furthermore, there was a significant difference in the WFA + -CSF1R (%) value between patients with cirrhosis who did not develop hepatocellular carcinoma and those with cirrhosis. 非HCC-LC患者におけるWFA+-CSF1R％値による発がん率（a）トレーニングセットでは、高WFA+-CSF1R％値患者のHCC発がん率は低値患者より高かった（P=0.006）。５年累積発がん率は高WFA+-CSF1R％値LC患者で75％、低WFA+-CSF1R％値LC患者の35％であった（p = 0.006）（b）バリデーションセットでは高WFA+-CSF1R％値患者のHCC発がん率は低値患者より高かった（P=0.005）。累積発がん率は高WFA+-CSF1R％値患者で70％、低WFA+-CSF1R％値患者の42％であった。Carcinogenicity by WFA + -CSF1R% in non-HCC-LC patients (a) In the training set, patients with high WFA + -CSF1R% had higher HCC carcinogenesis than those with low (P = 0.006). 5-year cumulative carcinogenic rate high WFA + -CSF1R% value of 75% in the LC patients, low WFA + -CSF1R% value was 35% of LC patients (p = 0.006) (b) in the validation set high WFA + -CSF1R % Patients with HCC had a higher incidence than those with low values (P = 0.005). Cumulative cancer rates were 70% for patients with high WFA + -CSF1R% and 42% for patients with low WFA + -CSF1R%. ログランク検定で求められた最小P値法による予測生存率の値をWFA+-CSF1R％の最適カットオフ値から310ng/mlに決定した。The predicted survival value by the minimum P-value method determined by the log rank test was determined to be 310 ng / ml from the optimal cutoff value of WFA + -CSF1R%. 非HCC-LC患者におけるWFA+-CSF1R値による生存率のカプラン・マイヤー分析（a）トレーニングセットでは、高WFA+-CSF1R値LC患者（＞ 310 ng/ml、8名）の1、3、5年生存率はそれぞれ88％、60％、45％、であり、低WFA+-CSF1R値の患者（＜ 310 ng/ml、48名）で94％、89％、74％であった（p = 0.010）。（b）バリデーションセットでは高WFA+-CSF1R値LC患者（＞ 310 ng/ml、10名）の1、3、5年生存率はそれぞれ100％、71％、43％、低WFA+-CSF1R値の患者（＜ 310 ng/ml、35名）100％、100％、100％（p ＜ 0.003）であった。Kaplan-Meier analysis of survival rates with WFA + -CSF1R levels in non-HCC-LC patients (a) In the training set, 1, 3, 5 of high WFA + -CSF1R levels LC patients ( > 310 ng / ml, 8 patients) Annual survival rates were 88%, 60%, and 45%, respectively, and 94%, 89%, and 74% in patients with low WFA + -CSF1R levels (<310 ng / ml, 48 patients) (p = 0.010). (B) In the validation set, high WFA + -CSF1R LC patients ( > 310 ng / ml, 10 patients) have 1, 3, and 5 year survival rates of 100%, 71%, and 43%, respectively, and low WFA + -CSF1R levels Of patients (<310 ng / ml, 35) were 100%, 100%, 100% (p <0.003). CSF1Rの糖鎖プロファイリング；各種のレクチンを含むマイクロアレイ上に本発明者らが合成した標準CSF1R（rCSF1R）（LDN+およびLDN-）をそれぞれアプライし，抗CSF1R抗体でアッセイした。LDN(+)rCSF1RはHEK293細胞、LDN(-)rCSF1Rはノックアウト細胞で発現させたrCSF1Rである。Sugar chain profiling of CSF1R: Standard CSF1R (rCSF1R) (LDN + and LDN-) synthesized by the present inventors was applied to a microarray containing various lectins, and assayed with an anti-CSF1R antibody. LDN (+) rCSF1R is rCSF1R expressed in HEK293 cells, and LDN (−) rCSF1R is expressed in knockout cells. CSF1R糖タンパク質（マウスミエローマNS0細胞由来）においてGlyco-Ridge法で確認された糖ペプチドおよび糖鎖付加位置。IGOT（131120CSF-RTL-Am＋GOT-dd10-35g-01）により同定。Glycopeptide and glycosylation position confirmed by Glyco-Ridge method in CSF1R glycoprotein (derived from mouse myeloma NS0 cells). Identified by IGOT (131120CSF-RTL-Am + GOT-dd10-35g-01). CSF1R糖タンパク質（標準CSF1R糖タンパク質）においてGlyco-Ridge法で確認された糖ペプチドおよび糖鎖付加位置。コアペプチド候補は予測されるトリプシン分解配列に、意図しない分解配列が含まれる場合がある。Glycopeptide and glycosylation position confirmed by Glyco-Ridge method in CSF1R glycoprotein (standard CSF1R glycoprotein). The core peptide candidate may include an unintended degradation sequence in the predicted trypsin degradation sequence. Glyco-Ridge法による標準CSF1R糖タンパク質の糖鎖構造（主要構造）の解析結果Results of Glyco-Ridge analysis of sugar chain structure (main structure) of standard CSF1R glycoprotein 抗CSF1Rモノクローナル抗体(culture sup)のウェスタンブロットによる生化学的評価Biochemical evaluation of anti-CSF1R monoclonal antibody (culture sup) by Western blot 各ドメインを欠損する標準CSF1R糖タンパク質分子Standard CSF1R glycoprotein molecule lacking each domain 抗CSF1Rモノクローナル抗体のWestern Blotによる抗原認識部位の解析Analysis of antigen recognition site by Western Blot of anti-CSF1R monoclonal antibody 抗CSF1Rモノクローナル抗体の予想される抗原認識部位Anticipated antigen recognition site of anti-CSF1R monoclonal antibody 抗体-抗体ELISA測定系による総CSF1R分子(Total CSF1R)の検出Detection of total CSF1R molecule (Total CSF1R) by antibody-antibody ELISA measurement system 抗体-WFAレクチン サンドイッチELISA測定系によるWFA+-CSF1R分子の検出Detection of WFA + -CSF1R molecule by antibody-WFA lectin sandwich ELISA system WFAレクチン-抗体 サンドイッチELISA測定系によるWFA+-CSF1R分子の検出Detection of WFA + -CSF1R molecule by WFA lectin-antibody sandwich ELISA system 抗体CSR-3を用いたWFAレクチン-抗体サンドイッチELISA測定系による、LacdiNAc糖鎖含有CSF1R糖タンパク質分子の検出（図中、LDN(+)rCSF1Rは、標準rCSF1R、LDN(-)rCSF1Rは、LDN欠損株産生rCSF1R、NS0 rCSF1Rは、市販rCSF1R（R＆D社）である。）Detection of LacdiNAc sugar chain-containing CSF1R glycoprotein molecule by WFA lectin-antibody sandwich ELISA system using antibody CSR-3 (In the figure, LDN (+) rCSF1R is standard rCSF1R, LDN (-) rCSF1R is LDN-deficient (The strain-produced rCSF1R and NS0 rCSF1R are commercially available rCSF1R (R & D).) 抗体-WFAレクチン サンドイッチELISA測定系による、LacdiNAc糖鎖含有CSF1R糖タンパク質分子の検出（図中、LDN(+)rCSF1Rは標準rCSF1R、LDN(-)rCSF1RはLDN欠損株産生rCSF1R、NS0 rCSF1Rは市販rCSF1R（R＆D社）、NHSは健常人血清試料である。）Detection of LacdiNAc sugar chain-containing CSF1R glycoprotein molecule by antibody-WFA lectin sandwich ELISA system (R & D), NHS is a healthy human serum sample.) 抗体CSR-3を用いた抗体-WFAレクチン サンドイッチELISA測定系（上段）および抗体CSR-3を用いた抗体-抗体サンドイッチELISA測定系（下段）（図中、LDN(+)rCSF1Rは、標準rCSF1R、LDN(-)rCSF1Rは、LDN欠損株産生rCSF1R、NHSは健常人血清試料である。）Antibody-WFA lectin sandwich ELISA measurement system using antibody CSR-3 (upper) and antibody-antibody sandwich ELISA measurement system using antibody CSR-3 (lower) (in the figure, LDN (+) rCSF1R is standard rCSF1R, LDN (−) rCSF1R is an LDN-deficient strain-producing rCSF1R, and NHS is a healthy human serum sample.) 抗体CSR-3を用いた抗体-VVAレクチン サンドイッチELISA測定系（図中、LDN(+)rCSF1Rは、標準rCSF1R、LDN(-)rCSF1Rは、LDN欠損株産生rCSF1R、NHSは健常人血清試料である。）Antibody-VVA lectin sandwich ELISA measurement system using antibody CSR-3 (In the figure, LDN (+) rCSF1R is standard rCSF1R, LDN (-) rCSF1R is LDN-deficient strain produced rCSF1R, NHS is a healthy human serum sample .) 実施例８で作製した抗CSF1R抗体（CSR-3、CSR-4、CSR-18、CSR-21及びCSR-30抗体）を用いたELISA系がHCV肝炎由来肝細胞がん患者のpool血清を、健常人のpool血清と識別できたことを示す。ELISA system using anti-CSF1R antibody (CSR-3, CSR-4, CSR-18, CSR-21 and CSR-30 antibody) prepared in Example 8 is used for pool serum of HCV hepatitis-derived hepatocellular carcinoma patients, It shows that it could be distinguished from the pool serum of healthy people. 他の抗CSF1R抗体（CSR-3、CSR-4、CSR-5、CSR-6、CSR-18抗体）も肝細胞がん患者血清pool血清と健常人のpool血清とを識別できることを示す。It shows that other anti-CSF1R antibodies (CSR-3, CSR-4, CSR-5, CSR-6, CSR-18 antibodies) can also distinguish hepatoma serum sera from healthy human pool sera. 抗体CSR-18を用いた抗体-VVAレクチン サンドイッチELISA測定系。WFAと同様に、VVAも抗CSF1R抗体と共にELISA系で肝細胞がん患者血清pool血清（K1、K2、K3）と健常人のpool血清とを識別できることを示す。Antibody-VVA lectin sandwich ELISA system using antibody CSR-18. Similar to WFA, VVA can be distinguished from serum pool serum (K1, K2, K3) of patients with hepatocellular carcinoma and pool serum of healthy individuals by ELISA together with anti-CSF1R antibody. 同じ濃度（の希釈系列）に調製したrCSF1R (LDN+)およびrCSF1R (LDN-)を用いて、抗CSF1R抗体-抗CSF1R抗体でのサンドイッチELISAによる全CSF1R測定を行った。それぞれバッファー系（BSA希釈液：Ａ）あるいは血清系（10%NHS希釈液：Ｂ）での検出を行った。その結果、CSF1R (LDN+)およびrCSF1R (LDN-)のいずれも、量（反応性）はほぼ同じように検出されている。Total CSF1R measurement was performed by sandwich ELISA with anti-CSF1R antibody-anti-CSF1R antibody using rCSF1R (LDN +) and rCSF1R (LDN-) prepared to the same concentration (dilution series). Detection was performed in a buffer system (BSA dilution: A) or a serum system (10% NHS dilution: B), respectively. As a result, the amount (reactivity) of both CSF1R (LDN +) and rCSF1R (LDN-) is detected in substantially the same manner. 抗体-抗体でのサンドイッチELISAによる全CSF1R測定を行った（Total）。同じ濃度（の希釈系列）に調製したrCSF1R (LDN+)およびrCSF1R (LDN-)を用いて、抗体-各共通糖鎖プローブレクチンでのサンドイッチELISAによる全CSF1R測定を行った（各レクチン名表記）。また、疾患特異的なCSF1R分子を、抗体-WFAレクチンでのサンドイッチELISAあるいは抗体-VVAレクチンでのサンドイッチELISAによって測定した（WFAあるいはVVA表記）。rCSF1R (LDN+)およびrCSF1R (LDN-)に対する、全CSF1R中のWFA陽性分子の相対比率（WFAシグナル値／共通糖鎖結合レクチンシグナル値）を算出してグラフ化した結果を示す。Total CSF1R was measured by antibody-antibody sandwich ELISA (Total). Using rCSF1R (LDN +) and rCSF1R (LDN-) prepared to the same concentration (dilution series), total CSF1R measurement was performed by sandwich ELISA using antibody-each common sugar chain probe lectin (each lectin name notation). In addition, disease-specific CSF1R molecules were measured by sandwich ELISA with antibody-WFA lectin or sandwich ELISA with antibody-VVA lectin (WFA or VVA notation). The graph shows the results of calculating and graphing the relative ratio (WFA signal value / common sugar chain-binding lectin signal value) of WFA-positive molecules in all CSF1R to rCSF1R (LDN +) and rCSF1R (LDN-). 抗体-抗体でのサンドイッチELISAによる全CSF1R測定を行った（Total）。また、同じ濃度（の希釈系列）に調製したrCSF1R (LDN+)およびrCSF1R (LDN-)を用いて、抗体-各共通糖鎖プローブレクチンでのサンドイッチELISAによる全CSF1R測定を行った（各レクチン名表記）。また、疾患特異的なCSF1R分子を、抗体-WFAレクチンでのサンドイッチELISAあるいは抗体-VVAレクチンでのサンドイッチELISAによって測定した（WFAあるいはVVA表記）。その後、吸光度シグナル値より相対的な濃度値として補正を行った。この補正濃度値を用いて、rCSF1R (LDN+)およびrCSF1R (LDN-)に対する、全CSF1R中のWFA陽性分子の相対比率（WFA-CSF1R濃度/共通糖鎖結合レクチン反応性CSF1R濃度）を算出してグラフ化した結果を示す。Total CSF1R was measured by antibody-antibody sandwich ELISA (Total). In addition, rCSF1R (LDN +) and rCSF1R (LDN-) prepared at the same concentration (dilution series) were used to measure all CSF1R by sandwich ELISA with antibody-common sugar chain probe lectins (each lectin name notation) ). In addition, disease-specific CSF1R molecules were measured by sandwich ELISA with antibody-WFA lectin or sandwich ELISA with antibody-VVA lectin (WFA or VVA notation). Thereafter, the concentration was corrected as a relative concentration value from the absorbance signal value. Using this corrected concentration value, calculate the relative ratio of WFA-positive molecules (WFA-CSF1R concentration / common glycan-binding lectin-reactive CSF1R concentration) in all CSF1R to rCSF1R (LDN +) and rCSF1R (LDN-) The graphed result is shown. 同じ濃度（の希釈系列）に調製したrCSF1R (LDN+)およびrCSF1R (LDN-)を用いて、抗体-VVAレクチンでのサンドイッチELISAによる測定を行った。それぞれバッファー系（BSA希釈液：Ａ）あるいは血清系（10%NHS希釈液：Ｂ）での検出を行った。その結果、抗体-WFAレクチンでのサンドイッチELISAの場合（図２１）と同じように、rCSF1R(LDN+)が濃度依存に反応性が上昇しているのに比較して、rCSF1R(LDN-)では反応性が殆ど上がらない。また、この結果はバッファー系でも血清系でも変わらないことを示す。Using rCSF1R (LDN +) and rCSF1R (LDN-) prepared to the same concentration (dilution series), measurement by sandwich ELISA with antibody-VVA lectin was performed. Detection was performed in a buffer system (BSA dilution: A) or a serum system (10% NHS dilution: B), respectively. As a result, as in the sandwich ELISA with antibody-WFA lectin (FIG. 21), the reactivity of rCSF1R (LDN +) increased compared to that of rCSF1R (LDN +), depending on the concentration. Sex is hardly improved. This result also shows that there is no change in the buffer system or the serum system. 同じ濃度（の希釈系列）に調製したrCSF1R (LDN+)およびrCSF1R (LDN-)を用いて、抗体-各共通糖鎖プローブレクチンでのサンドイッチELISAによる全CSF1R測定を行った。LEL（Ａ，Ｂ）、STL（Ｃ，Ｄ）， TJA-I（Ｅ，Ｆ）での検出を行った。それぞれバッファー系（BSA希釈液：Ａ，Ｃ，Ｅ）あるいは血清系（10%NHS希釈液：Ｂ，Ｄ，Ｆ）での検出を行った。その結果、全CSF1R量はほぼ同じように検出されている。Using rCSF1R (LDN +) and rCSF1R (LDN-) prepared to the same concentration (dilution series), total CSF1R measurement was performed by sandwich ELISA with antibody-each common sugar chain probe lectin. Detection was performed with LEL (A, B), STL (C, D), and TJA-I (E, F). Detection was performed in a buffer system (BSA dilution: A, C, E) or serum system (10% NHS dilution: B, D, F), respectively. As a result, the total CSF1R amount is detected almost in the same manner.
１．肝硬変の病態について
　我が国における肝硬変の患者数は20～25万人と推定され、成因としては80％近くが肝炎ウイルスで、特にC型肝炎ウイルス（HCV）（62.3％）によるウイルス性慢性肝炎に起因する場合が多い。慢性肝炎から肝硬変に至る病態は、肝臓のグリソン領域及び肝小葉に出現する線維性変化を病理形態学的に捉えて、軽度（F1）、中度（F2）、重度（F3）、肝硬変期（F4）に分類される（新犬山式分類方法）。
　本発明において、「肝硬変」というとき、肝臓の線維化の程度がF4に相当する状態にある場合を指す。病態としては、肝細胞の減少による肝機能不全及び門脈圧の亢進による食道胃静脈瘤の形成などがあげられ、重症化すると黄疸、肝性脳症、腹水貯留、消化管出血などがみられる。そして、年率7％程度の確率で肝細胞がんが出現する。
　本発明は，このような重篤な肝疾患である肝硬変患者における体液（血清など）試料を用いて肝細胞がん発症リスクを予測するための「肝細胞がん発症リスク指数」及び肝細胞がんを発症していない肝硬変患者の予後（生存率）を予測するための「肝硬変予後予測指数」を提供することにある。 1. About the pathophysiology of cirrhosis The number of patients with cirrhosis in Japan is estimated to be 250,000 to 250,000, and the cause is nearly 80% due to hepatitis virus, especially due to viral hepatitis due to hepatitis C virus (HCV) (62.3%) There are many cases to do. The pathophysiology from chronic hepatitis to cirrhosis is based on the pathological morphology of fibrotic changes in the liver Gleason region and liver lobule, and is mild (F1), moderate (F2), severe (F3), liver cirrhosis ( F4) (New Inuyama classification method).
In the present invention, “cirrhosis” refers to a case where the degree of liver fibrosis is in a state corresponding to F4. The pathological conditions include hepatic dysfunction due to decreased hepatocytes and the formation of esophageal gastric varices due to increased portal pressure, and severe cases include jaundice, hepatic encephalopathy, ascites retention, and gastrointestinal bleeding. And hepatocellular carcinoma appears with a probability of about 7% per year.
The present invention relates to a “hepatocellular carcinoma onset risk index” and a hepatocyte for predicting the risk of developing hepatocellular carcinoma using a body fluid (serum etc.) sample in a cirrhotic patient who is such a serious liver disease. Is to provide a “cirrhosis prognostic index” for predicting the prognosis (survival rate) of patients with cirrhosis who have not developed cancer.
（定義）
　本明細書において「被験者」とは、検査に供される者、すなわち後述する試料を提供する者を指し、肝硬変患者、好ましくは肝細胞がんに罹患していない肝硬変患者である。または、被験者が、肝疾患（急性肝炎、慢性肝炎、肝線維化、肝硬変）に罹患している患者、肝細胞がん患者又は健常者である場合を含む。
　「試料」とは、前記被験者から採取され、本実施形態の判定方法に供される体液であって、「体液」とは、被験者から採取された液体状の生体試料をいう。例えば、血液（血清、血漿及び間質液を含む）、胆汁、リンパ液、組織液（組織間液、細胞間液）、体腔液、各組織若しくは細胞の抽出液、胸水、痰、髄液、涙液、鼻汁、唾液、尿、膣液、***等が挙げられる。好ましくは、より好ましくは血清、血漿、胆汁である。典型的には血清試料であるため、本明細書では主として血清試料について述べる。体液は、被験者から採取したものを必要に応じて希釈若しくは濃縮、又はヘパリンのような血液凝固阻止剤を添加する等の処理を行なった後に使用してもよいし、そのような前処理を行なうことなく、そのまま使用してもよい。体液の採取は、当該分野の公知の方法に基づいて行なえばよい。例えば、血液やリンパ液であれば、公知の採血方法に従えばよい。具体的には、末梢血であれば、末梢部の静脈等に注射をして採取することができる。体液は、採取後直ちに利用してもよいし、冷凍又は冷蔵により一定期間保存した後、必要に応じて解凍等の処理を行ない利用することもできる。本実施形態において、血清を用いる場合には、10μL～100μL、20μL～80μL、30μL～70μL、40μL～60μL又は45μL～55μLの容量を用いれば、十分量の肝硬変患者の肝細胞がん発症リスク又は予後を定量的に予測判定するために必要な、WFAに結合性を示すCSF1R分子（WFA+-CSF1R）の値、及び全CSF1R量を測定することができる。 (Definition)
In the present specification, the “subject” refers to a person who is subjected to a test, that is, a person who provides a sample to be described later, and is a cirrhosis patient, preferably a cirrhosis patient not suffering from hepatocellular carcinoma. Alternatively, the subject includes a patient suffering from liver disease (acute hepatitis, chronic hepatitis, liver fibrosis, cirrhosis), a hepatocellular carcinoma patient, or a healthy person.
The “sample” is a body fluid collected from the subject and used for the determination method of the present embodiment, and the “body fluid” refers to a liquid biological sample collected from the subject. For example, blood (including serum, plasma and interstitial fluid), bile, lymph fluid, tissue fluid (tissue fluid, intercellular fluid), body cavity fluid, extract of each tissue or cell, pleural effusion, sputum, cerebrospinal fluid, tear fluid Nasal discharge, saliva, urine, vaginal fluid, semen and the like. More preferred are serum, plasma and bile. Since it is typically a serum sample, the present description mainly refers to serum samples. The body fluid may be used after performing treatment such as dilution or concentration of a sample collected from a subject, addition of a blood coagulation inhibitor such as heparin as necessary, or such pretreatment. It may be used as it is. The body fluid may be collected based on a known method in the field. For example, in the case of blood or lymph, a known blood collection method may be followed. Specifically, in the case of peripheral blood, it can be collected by injection into a peripheral vein or the like. The bodily fluid may be used immediately after collection, or may be used after being frozen or refrigerated for a certain period of time and then subjected to processing such as thawing as necessary. In the present embodiment, when serum is used, a sufficient amount of hepatoma can develop in patients with cirrhosis or cirrhosis if a volume of 10 μL to 100 μL, 20 μL to 80 μL, 30 μL to 70 μL, 40 μL to 60 μL, or 45 μL to 55 μL is used. The value of CSF1R molecule (WFA + -CSF1R) that binds to WFA and the total amount of CSF1R necessary for quantitatively predicting prognosis can be measured.
２．肝硬変患者の肝細胞がん発症リスク指数及び予後予測指数について
（２－１）肝硬変患者の肝細胞がん発症リスク指数
　本発明の肝硬変患者に対する発がんリスク又は予後予測方法においては、体液（血清）中のWFA及び／又はVVAに結合性を示す糖鎖を含有するCSF1R分子の量（WFA+-CSF1R）の測定が重要である。しかし、（WFA+-CSF1R）の値は、後述のように肝硬変患者の予後予測マーカーと呼ぶことはできるとしても、肝硬変患者の肝細胞がんの発がんリスクを予測する数値とはならない。肝硬変患者の肝細胞がんの発がんリスクを予測するためには、全CSF1R中に占める（WFA+-CSF1R）の割合（WFA+-CSF1R％）を算出する必要がある。
　なお、本発明では、肝硬変患者の肝細胞がんの発症リスクを予測するための「全CSF1R中に占める（WFA+-CSF1R）の割合」を、「％」で表記しているが、両者の割合を示す表記法であれば、小数点表記、分数表記などであってもかまわない。例えば、100%を1としたときの小数又は分数での表記（あるいは全CSF1R量を1とした時の、全CSF1R量に対するWFA結合性糖鎖含有CSF1R量を相対比として表した数値の表記）であったり、千分率（‰）で表記した値でも良い。
　具体的には、肝硬変患者の発がんリスクの判定には、下記の肝細胞がん発症リスク指数値（WFA+-CSF1R％）を算出して判定する。
　肝硬変患者の肝細胞がん発症リスク指数（WFA+-CSF1R％）
＝「WFA結合性糖鎖含有CSF1R量／全CSF1R量×100（％）」又は
＝「WFA結合性糖鎖含有CSF1R量／CSF1R特異的レクチン結合性糖鎖含有CSF1R量×100（％）」
　なお、ここで、「CSF1R特異的レクチン」とは、健常人と肝硬変患者の体液（血清）中のCSF1R上の糖鎖への反応性において差が無いものをいう。つまり体液（血清）中の全CSF1Rタンパク質上の糖鎖に対して反応性を有するレクチンを意味する。典型的には、非特許文献１、Fig3Bに示されたRCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA等のレクチンが相当する。 2. About hepatocellular carcinoma risk index and prognostic index for cirrhosis patients (2-1) Hepatocellular carcinoma risk index for cirrhosis patients In the method for predicting cancer risk or prognosis for cirrhosis patients according to the present invention, It is important to measure the amount of CSF1R molecule (WFA + -CSF1R) containing a sugar chain that binds to WFA and / or VVA. However, although the value of (WFA + -CSF1R) can be called a prognostic marker for cirrhosis patients as described later, it does not predict the risk of developing hepatocellular carcinoma in cirrhosis patients. In order to predict the risk of developing hepatocellular carcinoma in patients with cirrhosis, it is necessary to calculate the ratio (WFA + -CSF1R%) of all CSF1R (WFA + -CSF1R).
In the present invention, the “ratio of (WFA + -CSF1R) in the total CSF1R” for predicting the risk of developing hepatocellular carcinoma in cirrhosis patients is expressed in “%”. As long as it is a notation showing a ratio, it may be a decimal notation or a fraction notation. For example, a decimal or fractional representation when 100% is 1 (or a numerical representation of the CSF1R containing WFA-binding sugar chain as a relative ratio to the total CSF1R when the total CSF1R is 1) Or a value expressed in thousandths (‰).
Specifically, the risk of carcinogenesis in cirrhosis patients is determined by calculating the following hepatocellular carcinoma risk index (WFA + -CSF1R%).
Hepatoma risk index for patients with cirrhosis (WFA + -CSF1R%)
= “WFA-binding sugar chain-containing CSF1R amount / total CSF1R amount × 100 (%)” or “WFA-binding sugar chain-containing CSF1R amount / CSF1R-specific lectin-binding sugar chain-containing CSF1R amount × 100 (%)”
Here, “CSF1R-specific lectin” refers to those that have no difference in reactivity to sugar chains on CSF1R in body fluid (serum) of healthy individuals and cirrhotic patients. In other words, it means a lectin that is reactive to sugar chains on all CSF1R proteins in body fluid (serum). Typically, lectins such as RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA shown in Non-Patent Document 1 and FIG.
　後述のように、WFA結合性糖鎖含有CSF1R量と言うときの、「WFA結合性糖鎖」を含有するCSF1Rは、WFAのみならずVVAレクチンとも特異的に結合するが、このことは、CSF1Rがこれらのレクチンにそれぞれ認識されうる多くの糖鎖構造群のうち、共通した、あるいは似たようなエプトープ構造部分を有する糖鎖構造を含有する可能性が高い。それはいくつかの構造があると考えられるが、本発明の他の実験結果からも、その一つが「LDN糖鎖」であることが強く示唆される。しかし、 WFAが結合する糖鎖とVVAが結合する糖鎖が、同一CSF1R分子上の別々の部位に結合している可能性もないとは言い切れないことを踏まえて「WFA結合性糖鎖」として着目するCSF1R上の糖鎖を「WFA及び／又はVVA（以下、「WFA／VVA」と表記する。）結合性糖鎖」と称し、「WFA結合性糖鎖含有CSF1R量」を、「WFA／VVA結合性糖鎖含有CSF1R量」とも表す。
　そして、本発明の、肝硬変患者の肝細胞がんの発症リスク値の算出方法は、
肝硬変患者由来の被験者から採取された一定容量の体液試料（単に被検試料ともいう。）の全CSF1R量（Ａ）及びWFA／VVA結合性糖鎖含有CSF1R量（Ｂ）を測定し、両者の測定値から、全CSF1Rに占めるWFA／VVA結合性糖鎖含有CSF1R量の比率（Ｃ）を「Ｃ（％）＝（Ｂ）／（Ａ）×100」として算出する方法である。 As described later, when referring to the amount of CSF1R containing a WFA-binding sugar chain, CSF1R containing a `` WFA-binding sugar chain '' specifically binds not only to WFA but also to a VVA lectin. Among the many sugar chain structure groups that can be recognized by these lectins, there is a high possibility that they contain a sugar chain structure having a common or similar apoptotic structure portion. It is thought that there are several structures, but other experimental results of the present invention strongly suggest that one of them is an “LDN sugar chain”. However, in light of the fact that there is no possibility that the sugar chain to which WFA binds and the sugar chain to which VVA binds bind to different sites on the same CSF1R molecule, The sugar chain on CSF1R to be noted as “WFA and / or VVA (hereinafter referred to as“ WFA / VVA ”) binding sugar chain” is referred to as “WFA binding sugar chain-containing CSF1R amount” as “WFA / VVA-binding sugar chain-containing CSF1R amount ".
And the calculation method of the onset risk value of hepatocellular carcinoma of cirrhosis patients of the present invention,
Measure the total CSF1R amount (A) and WFA / VVA-binding sugar chain-containing CSF1R amount (B) of a certain volume of body fluid sample (also referred to simply as a test sample) collected from a subject derived from a cirrhotic patient. This is a method of calculating the ratio (C) of the amount of CSF1R containing WFA / VVA-binding sugar chains to the total CSF1R from the measured value as “C (%) = (B) / (A) × 100”.
　本発明の肝細胞がん発症リスク指数（WFA+-CSF1R％）は、あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者のWFA+-CSF1R％を測定しておき、患者をフォローアップして、肝細胞がん発症の有無を、例えばログランク検定で決定した最小P値法に適用し、上下10%を除外し発がん率の最適カットオフ値を求めることで、正確に求めることができる。
　なお、十分な母数というとき、統計的には母集団が大きくなるほど正確になるが、母集団を確保するための時間、労力を考えると、一般的には、数十例から数百例である。数百から1000例以上あるとより好ましい。すなわち、本発明で「十分な母数」とは、10～6000例、10～5000例、10～4000例、10～3000例、10～1000例、好ましくは30～3000例、30～2000例、30～1000例、より好ましくは40～2000例、40～1000例、50～2000例、50～1000例、さらに好ましくは50～500例、100～500例を指す。
　肝硬変患者にとって、発がんリスク指数（WFA+-CSF1R％）の最適カットオフ値以上では発がん率がきわめて高いと予測できる。
　本実施例では、Training set及びValidation setあわせて101名の母数団で求めたところ35.0％となり、高値群で5年累積発現率が75％、低値群で30～42％と有意に高い（P＝0.005～0.006）ことが証明された。
　母数がさらに大きくなれば、若干この35.0％の値は変動するかもしれないが、本実施例の結果からみて、肝硬変患者における肝細胞がん発症リスク指数の最適カットオフ値は、30.0～40.0％、少なくとも25.0～45.0％の範囲内であるということができる。すなわち、肝硬変患者における肝細胞がん発症リスク指数（WFA+-CSF1R％）が35.0±10.0％以上、好ましくは35.0±5.0％以上である場合に、有意に肝細胞がんを発症すると判定できる。反対に、35.0±10.0％未満、好ましくは35.0±5.0％未満であれば肝細胞がんが発症する確率は有意に低いと判定できる。 Hepatocellular carcinoma risk index of the present invention (WFA + -CSF1R%) is measured beforehand the WFA + -CSF1R% of pre sufficient population parameter of hepatocellular carcinoma unaffected cirrhosis patients, and follow-up of patients The presence or absence of hepatocellular carcinoma can be accurately determined by applying the minimum P-value method determined by the log rank test, for example, and excluding the upper and lower 10% to determine the optimal cut-off value of the carcinogenic rate .
In addition, when it is said that there is a sufficient population, statistically, the more accurate the population, the more accurate it will be. However, considering the time and effort required to secure the population, in general, there are several tens to hundreds of cases. is there. It is more preferable that there are several hundred to 1000 cases or more. That is, in the present invention, “sufficient parameters” means 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 1000, preferably 30 to 3000, 30 to 2000. 30-1000 examples, more preferably 40-2000 examples, 40-1000 examples, 50-2000 examples, 50-1000 examples, more preferably 50-500 examples, 100-500 examples.
For patients with cirrhosis, the carcinogenic risk index (WFA + -CSF1R%) can be predicted to be extremely high at or above the optimal cutoff value.
In this example, the training set and validation set for the 101 population were 35.0%. The 5-year cumulative incidence was 75% in the high-value group and 30-42% in the low-value group. (P = 0.005-0.006) proved.
The value of 35.0% may fluctuate slightly as the population increases further. However, from the results of this example, the optimal cutoff value for hepatocellular carcinoma risk index in patients with cirrhosis is 30.0 to 40.0. %, At least in the range of 25.0-45.0%. That is, when the hepatoma risk index (WFA + -CSF1R%) in cirrhosis patients is 35.0 ± 10.0% or more, preferably 35.0 ± 5.0% or more, it can be determined that hepatocellular carcinoma is significantly developed. On the other hand, if it is less than 35.0 ± 10.0%, preferably less than 35.0 ± 5.0%, it can be determined that the probability of developing hepatocellular carcinoma is significantly low.
　なお、定量的検査について、検査の陽性、陰性を分ける値のことをカットオフ値と呼ぶ。カットオフ値は、ある疾患に罹患した患者群（対象群A）と非患者群（対象群B）とを分ける値である。これらは測定対象となる母集団の規模（数）によって多少の変動をすると考えられるが、統計学的に常法とされる計算方法に依れば、自ずと最適なカットオフ値を設定することが出来る。本発明の実施態様では、上述の汎用的な方法として、ログランク検定で決定した最小P値法に適用し、上下10%を除外し発がん率の最適カットオフ値を計算したが、後述の他の方法を用いることもできる。一般に患者群と非患者群の検査値分布が重ならないのであれば、互いの検査値分布の範囲に含まれないような中間値をカットオフ値として設定することも出来る。一方、検査値分布の範囲が重複するのであれば、最適なカットオフ値はROC曲線（Receiver Operator Characteristic Curve）などを用いて設定することも出来る。ROC曲線は縦軸に感度、横軸に偽陽性率（＝1-特異度）をとって、カットオフ値を変動させながらプロットしたときに得られる曲線である。ROC曲線におけるカットオフ値の決め方としては、以下が挙げられる。感度と特異度の優れた独立変数のROC曲線は、左上隅に近づいていくという事実から、この左上隅との距離が最小となる点をカットオフ値にすることができる。また、Youden index（「感度+特異度-1」が最大値となるポイントをYouden indexと呼ぶ）を用いた方法でもカットオフ値を算出できる。最も予測能・診断能が低い独立変数のROC曲線、すなわちAUC = 0.500となる斜点線から最も離れたポイントをカットオフ値にする。すなわち、（感度+特異度-1）を計算して、その最大値となるポイントをカットオフ値にすることができる。さらには、Cox回帰法で決定した最小P値法に適用し、上下10%を除外して生存率の最適カットオフ値を求めることもできる。 For quantitative tests, the value that separates positive and negative tests is called the cut-off value. The cut-off value is a value that separates a patient group (subject group A) suffering from a certain disease from a non-patient group (subject group B). These are considered to vary slightly depending on the size (number) of the population to be measured, but depending on the calculation method that is statistically standard, it is possible to set an optimal cutoff value by itself. I can do it. In the embodiment of the present invention, the above-described general-purpose method is applied to the minimum P-value method determined by the log rank test, and the optimal cutoff value of the carcinogenic rate is calculated by excluding the upper and lower 10%. This method can also be used. In general, if the test value distributions of the patient group and the non-patient group do not overlap, an intermediate value that is not included in the range of the test value distribution can be set as the cutoff value. On the other hand, if the range of the inspection value distribution overlaps, the optimum cutoff value can be set using a ROC curve (Receiver Operator Characteristic Curve). The ROC curve is a curve obtained when plotting while varying the cutoff value with sensitivity on the vertical axis and false positive rate (= 1-specificity) on the horizontal axis. The following is mentioned as a method of determining the cutoff value in the ROC curve. Due to the fact that an independent variable ROC curve with excellent sensitivity and specificity approaches the upper left corner, the point at which the distance from the upper left corner is minimum can be set as a cutoff value. The cut-off value can also be calculated by a method using Youden index (a point where “sensitivity + specificity−1” is the maximum value is called Youden index). The ROC curve of the independent variable with the lowest predictive ability and diagnostic ability, that is, the point farthest from the oblique line where AUC と = 0.500 is set as the cut-off value. That is, (sensitivity + specificity-1) can be calculated, and the point having the maximum value can be set as a cutoff value. Furthermore, it can be applied to the minimum P-value method determined by the Cox regression method, and the optimal cutoff value of the survival rate can be obtained by excluding the upper and lower 10%.
（２－２）肝硬変患者の予後（生存率）予測指数
　本発明においては、WFA結合性糖鎖含有CSF1R量（WFA+-CSF1R値）の数値がそのままで肝細胞がんに罹患していない肝硬変患者の生存率と高い相関関係にあることを見いだし、WFA+-CSF1R値が肝硬変患者の予後因子であることを実証した。
　具体的には、肝硬変患者の予後（生存率）の予測は、下記の肝細胞がん予後判定指数（WFA+-CSF1R）を算出して判定することができる。
　肝硬変患者の予後判定指数＝WFA+-CSF1R ng/ml
　また、本発明の肝硬変患者における予後判定指数値の算出方法は、
肝硬変患者である被験者から採取された一定容量の体液試料（被検試料）中のWFA／VVA結合性糖鎖を含有するCSF1R量（Ｂ）を測定する工程で得られた「Ｂng/ml（WFA+-CSF1R ng/ml）」の値を、被験者の予後判定指数値とする方法である。 (2-2) Prognosis (survival rate) predictive index of cirrhosis patients In the present invention, cirrhosis not affecting hepatocellular carcinoma with the same value of the amount of CSF1R containing WFA-binding sugar chain (WFA + -CSF1R value) We found that it was highly correlated with patient survival and demonstrated that WFA + -CSF1R levels are a prognostic factor in patients with cirrhosis.
Specifically, the prediction of the prognosis (survival rate) of cirrhosis patients can be determined by calculating the following hepatocellular carcinoma prognosis determination index (WFA + -CSF1R).
Prognostic index for patients with cirrhosis = WFA + -CSF1R ng / ml
Further, the method for calculating the prognostic index value in the cirrhosis patient of the present invention,
“Bng / ml (WFA) obtained in the step of measuring the amount of CSF1R (B) containing a WFA / VVA-binding sugar chain in a body fluid sample (test sample) collected from a subject who is a cirrhotic patient. + -CSF1R ng / ml) ”is used as the prognostic index value of the subject.
　本発明の予後判定指数（WFA+-CSF1R ng/ml）は、あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者のWFA+-CSF1R ng/mlを測定しておき、患者を最低5年間フォローアップして、その生存の有無データをログランク検定で決定した最小P値法に適用し、上下10%を除外し生存率の最適カットオフ値を求めることで、正確に求めることができる。
　なお、本発明では、肝硬変患者の予後を予測するための指数を、「ng/ml」の単位で表記しているが、被験者の体液（血清）試料中に含まれる「WFA結合性糖鎖含有CSF1R量」を表す典型的な数値を示したものであるからmg/ml、w/v％などの他の単位で表記してもよく、また対数表示、あるいは特定の計算式（演算式）や係数によって換算された演算値の値など他の表記であってもかまわない。
　また、これらのカットオフ値よりカットオフインデックス（cut off index: C.O.I）を算出することも出来る。C.O.Iはカットオフ値に対する比率で求められ、1.0が判定の陽性と陰性の境界値となる。（一般的に、その基準範囲内に95％の検査結果が含まれると考えられている。）
　臨床検査では、むしろ測定値による値のブレを無くすなどのために、実際の値からカットオフインデックスを計算し、これを用いることが多い。
　本実施例では、ログランク検定で決定した最小P値法でWFA+-CSF1Rの最適カットオフ値310ng/mlを導き出し、時間依存的ROC曲線で生存率を検討したところ、WFA+-CSF1R値310ng/ml以上でHRが3.63（95％CI1.25-10.54，p=0.011）であり、肝硬変患者の累積生存率をカプラン・マイヤー解析で検討すると、WFA+-CSF1R値高値群は低値群に比較して有意に生存率が低いことが証明された。
　母数がさらに大きくなれば、若干310ng/mlの値は変動するかもしれないが、本実施例の結果からみて、肝硬変患者における予後判定指数の最適カットオフ値は、260～360ng/ml，少なくとも210～410ng/mlの範囲内であるということができる。すなわち、肝硬変患者における予後判定指数（WFA+-CSF1R ng/ml）が310±100ng/ml以上、好ましくは310±50ng/ml以上である場合に、有意に予後（生存率）が悪いと判定できる。反対に、310±100ng/ml未満、好ましくは310±50ng/ml未満であれば、有意に予後が良いと判定できる。
　この最適カットオフ値は、前述した他のカットオフ値の算出方法を用いて決定してもよい。 The prognostic index (WFA + -CSF1R ng / ml) of the present invention was determined in advance by measuring WFA + -CSF1R ng / ml of liver cirrhosis patients not affected by hepatocellular carcinoma with a sufficient population, and the patients were kept for at least 5 years. By following up and applying the existence presence / absence data to the minimum P-value method determined by the log rank test, excluding the upper and lower 10%, the optimum cutoff value of the survival rate can be obtained accurately.
In the present invention, the index for predicting the prognosis of cirrhosis patients is expressed in units of “ng / ml”. However, “WFA-binding sugar chain-containing” contained in the body fluid (serum) sample of the subject. Because it shows a typical numerical value representing CSF1R amount, it may be expressed in other units such as mg / ml, w / v%, logarithmic display, or a specific calculation formula (arithmetic formula) Other notations such as the value of the operation value converted by the coefficient may be used.
Further, a cut-off index (COI) can be calculated from these cut-off values. COI is calculated as a ratio to the cutoff value, and 1.0 is the boundary between positive and negative judgments. (In general, it is considered that 95% of the test results are included in the standard range.)
In clinical examinations, a cut-off index is often calculated from an actual value and used in order to eliminate the fluctuation of the value due to the measured value.
In this example, the optimal cutoff value of 310 ng / ml of WFA + -CSF1R was derived by the minimum P-value method determined by the log rank test, and the survival rate was examined by a time-dependent ROC curve. The WFA + -CSF1R value was 310 ng. When the cumulative survival rate of cirrhosis patients is examined by Kaplan-Meier analysis, the high WFA + -CSF1R level group is changed to the low level group with HR of 3.63 (95% CI1.25-10.54, p = 0.011) In comparison, the survival rate was proved to be significantly lower.
If the parameter is further increased, the value of 310 ng / ml may fluctuate slightly. However, from the results of this example, the optimal cutoff value of the prognostic index in patients with cirrhosis is 260 to 360 ng / ml, at least It can be said that it is in the range of 210 to 410 ng / ml. That is, when the prognostic index (WFA + -CSF1R ng / ml) in patients with cirrhosis is 310 ± 100 ng / ml or more, preferably 310 ± 50 ng / ml or more, it can be judged that the prognosis (survival rate) is significantly poor. . On the contrary, if it is less than 310 ± 100 ng / ml, preferably less than 310 ± 50 ng / ml, it can be judged that the prognosis is significantly better.
The optimum cutoff value may be determined using the other cutoff value calculation method described above.
（２－３）CSF1R及び体液（血清）中の全CSF1R量の測定
　CSF1Rとは、単球系の細胞の分化に必須なcolony stimulating factor 1 (CSF1)のレセプター（マクロファージ刺激因子-1受容体）であり、細胞表面に存在する。肝臓では主にKupffer細胞を含む単球系の細胞、肝星細胞、肝実質細胞で発現している事が知られている。CSF1Rは細胞の活性化とともに（活性化する）細胞外メタロプロテアーゼによって細胞外ドメインが切り出される事が知られており、血中CSF1Rは切り出された細胞外ドメインであると考えられる。972アミノ酸からなり（配列番号１，２）、従来から肝細胞がんマーカーとしても用いられていた。
　組換えCSF1Rは、例えば、Fc融合型（NS0）；R&D Systems社）など複数市販されている。今回CSF1R上のWFA結合性糖鎖の正確な糖鎖構造および糖鎖結合位置を特定するために、既知のCSF1R塩基配列（配列番号１）を利用してプライマーを設計し、ヒト単球性白血病細胞株（THP-1）由来cDNAを鋳型にCSF1R遺伝子をクローニングし、HEK293細胞を宿主として「標準組換えCSF1R」を製造した。標準組換えCSF1R上の糖鎖構造は、下記（２－４）に示す通りである（図１３）。 (2-3) Measurement of total CSF1R in CSF1R and body fluid (serum) CSF1R is a colony stimulating factor 1 (CSF1) receptor (macrophage stimulating factor-1 receptor) essential for the differentiation of monocyte cells And present on the cell surface. In the liver, it is known that it is expressed mainly in monocytic cells including Kupffer cells, hepatic stellate cells, and liver parenchymal cells. CSF1R is known to be cleaved by an extracellular metalloprotease with cell activation (activated), and blood CSF1R is considered to be a cleaved extracellular domain. It consists of 972 amino acids (SEQ ID NOs: 1 and 2) and has been conventionally used as a hepatocellular carcinoma marker.
A plurality of recombinant CSF1Rs are commercially available, for example, Fc fusion type (NS0); R & D Systems). In order to identify the exact sugar chain structure and sugar chain binding position of the WFA-binding sugar chain on CSF1R, a primer was designed using the known CSF1R base sequence (SEQ ID NO: 1), and human monocytic leukemia The CSF1R gene was cloned using a cell line (THP-1) -derived cDNA as a template, and “standard recombinant CSF1R” was produced using HEK293 cells as a host. The sugar chain structure on the standard recombinant CSF1R is as shown in (2-4) below (FIG. 13).
　試料中の全CSF1R量を検出するためには、抗CSF1Rモノクローナル抗体を用いることが好ましい（以下、単に「抗CSF1R抗体」ともいう。）。抗CSF1R抗体は、市販されている抗CSF1R抗体，例えば固相化抗体としてanti-CSF1R mAb Cat#MAB3292（R&D Systems社）や検出抗体（Total CSF1R用）としてbiotinylated anti-CSF1R pAb Cat#BAF329（R&D Systems社）などを使用可能であるが、CSF1Rを免疫原として、常法に従えば製造可能である。
　本発明では、抗CSF1Rモノクローナル抗体も新たに製造した。CSF1Rを免疫原として、常法に従い抗CSF1Rモノクローナル抗体を多数製造した。これらの抗CSF1Rモノクローナル抗体のうち、CSF1Rへの親和性の高い33クローンを選択し、直接的ELISAによるCSF1R 結合活性と共に、天然WFAレクチン又はsrWFAレクチンとのサンドイッチアッセイ系でのCSF1R上のWFA／VVA結合性糖鎖の検出性能を検証し、特に検出性能の高い複数の抗CSF1Rモノクローナル抗体をさらに選択した。そして、CSF1R上のWFA／VVA結合性糖鎖の検出性能の高い抗体のほとんどの認識ドメインは第２ドメイン又は第３ドメインに集中していることもわかった。なお、典型的な抗CSF1Rモノクローナル抗体産生ハイブリドーマについては、すでにNPMDに寄託している（ハイブリドーマCSR-3、CSR-4、CSR-18、CSR-21及びCSR-30を、それぞれ受領番号：NITE AP-02117～NITE AP-02121として寄託。その後受託番号：NITE P-02117～NITE P-02121が付与され、2016年9月7日付で国際寄託に移管されて、それぞれNITE BP-02117～NITE BP-02121が付与された。）。
　下記（表５）に示されるように、WFA／VVA-CSF1R抗体サンドイッチELISA系での検出に優れている抗体は認識ドメインには関係なく、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、CSR-29の抗体が優れており、特にCSR-3、CSR-4、CSR-18、CSR-21、CSR-30のWFA／VVA-結合性糖鎖含有CSF1R分子の検出能が高かった。 In order to detect the total amount of CSF1R in the sample, it is preferable to use an anti-CSF1R monoclonal antibody (hereinafter also simply referred to as “anti-CSF1R antibody”). Anti-CSF1R antibodies are commercially available anti-CSF1R antibodies, for example, anti-CSF1R mAb Cat # MAB3292 (R & D Systems) as a solid-phase antibody and biotinylated anti-CSF1R pAb Cat # BAF329 (R & D) as a detection antibody (for Total CSF1R). Systems) and the like can be used, but can be produced according to a conventional method using CSF1R as an immunogen.
In the present invention, an anti-CSF1R monoclonal antibody was also newly produced. A number of anti-CSF1R monoclonal antibodies were produced according to a conventional method using CSF1R as an immunogen. Among these anti-CSF1R monoclonal antibodies, 33 clones having high affinity for CSF1R were selected, and together with CSF1R binding activity by direct ELISA, WFA / VVA on CSF1R in a sandwich assay system with natural WFA lectin or srWFA lectin The detection performance of the binding sugar chain was verified, and a plurality of anti-CSF1R monoclonal antibodies with particularly high detection performance were further selected. It was also found that most recognition domains of antibodies with high ability to detect WFA / VVA-binding sugar chains on CSF1R are concentrated in the second domain or the third domain. Note that typical anti-CSF1R monoclonal antibody-producing hybridomas have already been deposited with NPMD (hybridomas CSR-3, CSR-4, CSR-18, CSR-21 and CSR-30, respectively, receipt number: NITE AP Deposited as -02117-NITE AP-02121, and then given the accession numbers: NITE P-02117-NITE P-02121, transferred to the international deposit on September 7, 2016, respectively, NITE BP-02117-NITE BP- 02121 was granted.).
As shown below (Table 5), antibodies excellent in detection by the WFA / VVA-CSF1R antibody sandwich ELISA system are CSR-3, CSR-4, CSR-18, CSR-, regardless of the recognition domain. 21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13, CSR-26, CSR-27, CSR-29 antibodies are excellent In particular, CSR-3, CSR-4, CSR-18, CSR-21, and CSR-30 were highly capable of detecting CSF1R molecules containing WFA / VVA-binding sugar chains.
（２－４）「CSF1R分子上のWFA及び／又はVVA（WFA／VVA）結合性糖鎖」
　肝硬変患者で検出される体液（血清）中の「CSF1R分子上のWFA結合性糖鎖」は、LacdiNAc構造を含む糖鎖（LDN糖鎖）であることが強く示唆される。当該LDN糖鎖を認識する化合物であれば，レクチン、抗体などどのような化合物であっても、CSF1R分子上のWFA／VVA結合性糖鎖量の測定に用いることができる可能性が高い。好ましくは、LDN糖鎖結合性レクチンであるWFA／VVAレクチンであり、天然のWFAレクチン、組換えWFAレクチン（配列番号４）、単量体組換えWFAレクチン（srWFAともいう。なお、図中では、単にrWFAと表記されている。）、VVAレクチンが好ましく、LDN糖鎖特異的に結合する単量体組換えWFAレクチンが最も好ましい。
　なお、肝硬変患者の体液（血清）中で増大するCSF1R上のWFAレクチン認識糖鎖構造が、非還元末端に「GalNAcβ1-4GlcNAcβ1-R」を有する糖鎖（LDN糖鎖）であることを強く示唆する実験として、本発明において製造された標準rCSF1R上の糖鎖構造および糖鎖位置の決定を行い、LDN糖鎖欠損株が産生するrCSF1RがWFAレクチンとの結合性を失う結果を得ている。 (2-4) “WFA and / or VVA (WFA / VVA) -binding sugar chain on CSF1R molecule”
It is strongly suggested that the “WFA-binding sugar chain on the CSF1R molecule” in body fluid (serum) detected in patients with cirrhosis is a sugar chain containing a LacdiNAc structure (LDN sugar chain). Any compound that recognizes the LDN sugar chain is likely to be used for the measurement of the amount of WFA / VVA-binding sugar chain on the CSF1R molecule, such as lectin and antibody. Preferably, it is a WFA / VVA lectin which is an LDN sugar chain-binding lectin, and is also referred to as a natural WFA lectin, a recombinant WFA lectin (SEQ ID NO: 4), or a monomeric recombinant WFA lectin (srWFA. VVA lectin is preferred, and monomeric recombinant WFA lectin that binds specifically to LDN sugar chains is most preferred.
In addition, it strongly suggests that the WFA lectin recognition sugar chain structure on CSF1R that increases in the body fluid (serum) of cirrhosis patients is a sugar chain (LDN sugar chain) with "GalNAcβ1-4GlcNAcβ1-R" at the non-reducing end. As an experiment, the sugar chain structure and sugar chain position on the standard rCSF1R produced in the present invention were determined, and rCSF1R produced by the LDN sugar chain-deficient strain lost the ability to bind to the WFA lectin.
＜WFAレクチン＞
　天然WFAは、マメ科のWisteria floribunda（ノダフジ）由来のレクチンであり、糖結合特異性としては、（末端）N-アセチルガラクトサミン（N-acetylgalactosamine：GalNAc）糖鎖を含むもの、特にLacdiNAc(LDN: GalNAc1-3GlcNAc-R)糖鎖構造に結合することが知られている。天然WFAは、配列番号４からなるWFAの全長からC末端側の13アミノ酸が失われたアミノ酸配列が二量体化した構造を有している（特許文献４）。
　また、通常の組換えWFA（配列番号４）は、二量体と単量体の混合物として得られ、LDN糖鎖結合性を含め、天然WFAと同一の糖鎖結合活性を有することが知られている（特許文献４）ので、組換えWFAも天然WFAと同様に本発明のWFAレクチンとして用いることができる。また、特許文献４中に記載されたWFA還元体などのWFA誘導体、その他WFA改変体もLDN結合活性を有しているため、同様に用いることができる。 <WFA lectin>
Natural WFA is a lectin derived from leguminous Wisteria floribunda (Nodafuji), and the sugar binding specificity includes (terminal) N-acetylgalactosamine (GalNAc) sugar chain, particularly LacdiNAc (LDN: GalNAc1-3GlcNAc-R) is known to bind to the sugar chain structure. Natural WFA has a structure in which the amino acid sequence in which 13 amino acids on the C-terminal side are lost from the full length of WFA consisting of SEQ ID NO: 4 is dimerized (Patent Document 4).
In addition, normal recombinant WFA (SEQ ID NO: 4) is obtained as a mixture of a dimer and a monomer, and is known to have the same sugar chain binding activity as natural WFA, including LDN sugar chain binding. Therefore, recombinant WFA can be used as the WFA lectin of the present invention in the same manner as natural WFA. Moreover, since WFA derivatives, such as a WFA reductant described in Patent Document 4, and other WFA variants have LDN binding activity, they can be used in the same manner.
＜単量体組換えWFAレクチン（srWFA）：LDN特異的結合性レクチン＞
　本発明者らが、以前に組換えWFA遺伝子（配列番号３）をクローニングし、C末端側のS-S結合形成を阻止する改変を行って単量体化したWFAレクチン（srWFA）であり、非還元末端に「GalNAcβ1-4GlcNAcβ1-R」を有する糖鎖（LDN糖鎖）特異的に結合する（特許文献４）。本実施例で実際に使用した「srWFA」は、そのうち配列番号３の272位の位置のCysをAlaに改変させることで単量体化し、さらに糖鎖結合活性に必要のないN結合型糖鎖付加部位146位のAsnをGlnに変異を導入して、酵母で大量生産したものである。 <Monomer Recombinant WFA Lectin (srWFA): LDN Specific Binding Lectin>
The present inventors have previously cloned a recombinant WFA gene (SEQ ID NO: 3) and modified it to prevent formation of an SS bond on the C-terminal side to form a monomer, which is a non-reducing WFA lectin (srWFA). It specifically binds to a sugar chain (LDN sugar chain) having “GalNAcβ1-4GlcNAcβ1-R” at its terminal (Patent Document 4). The “srWFA” actually used in this example was monomerized by modifying Cys at position 272 of SEQ ID NO: 3 to Ala, and further an N-linked sugar chain that is not required for sugar chain-binding activity. Asn at position 146 was introduced into Gln and a large amount was produced in yeast.
＜VVAレクチン＞
　VVAレクチンは、マメ科のVicia villosa (Hairy Vetch) seeds （ケヤハズエンドウ，ビロードクサフジ，ヘアリーベッチ）由来のレクチンであり、分子量は102 kDa ～ 144 kDaの糖タンパク質である。糖結合特異性としては、（末端）N-アセチルガラクトサミン（N-acetylgalactosamine：GalNAc）に結合することが知られている。 <VVA lectin>
VVA lectin is a lectin derived from the leguminous Vicia villosa (Hairy Vetch) seeds, and is a glycoprotein having a molecular weight of 102 kDa to 144 kDa. As sugar-binding specificity, it is known to bind to (terminal) N-acetylgalactosamine (GalNAc).
（２－５）体液中の「全CSF1R量」に代替できる「CSF1R特異的レクチン結合性糖鎖含有CSF1R量」の測定について
　前述のように、「CSF1R特異的レクチン」とは、「CSF1R恒常的糖鎖構造結合性レクチン」でもあり、健常人であるか肝硬変患者であるかにはかかわらず、体液（血清）中の全てのCSF1Rが恒常的に含有している糖鎖に対して反応性を有するレクチンである。
　したがって、体液試料中の「全CSF1R量」を直接測定する代わりに、体液試料中のCSF1R上の「CSF1R特異的レクチン」反応性糖鎖の量を測定することで、「全CSF1R量」が測定できる。すなわち、体液試料中のCSF1R上の「WFA及び／又はVVA結合性糖鎖」と「CSF1R特異的レクチン結合性糖鎖」との含有量の比率を求めることで、肝硬変患者における肝細胞がん発症リスク（WFA+-CSF1R％）を算出できる。 (2-5) Measurement of “CSF1R-specific lectin-binding sugar chain-containing CSF1R amount” that can be substituted for “total CSF1R amount” in body fluid As described above, “CSF1R-specific lectin” It is also a “glycan structure-binding lectin”, which is reactive to all CSF1R constitutively contained in body fluid (serum) regardless of whether it is a healthy person or a patient with cirrhosis. It has a lectin.
Therefore, instead of directly measuring the “total CSF1R amount” in the body fluid sample, the “total CSF1R amount” is measured by measuring the amount of the “CSF1R-specific lectin” reactive sugar chain on the CSF1R in the body fluid sample. it can. That is, by determining the content ratio of “WFA and / or VVA-binding sugar chain” and “CSF1R-specific lectin-binding sugar chain” on CSF1R in a body fluid sample, hepatocellular carcinoma in liver cirrhosis patients Risk (WFA + -CSF1R%) can be calculated.
　「CSF1R特異的レクチン」として用いることができる典型的なレクチンは、非特許文献１、Fig3Bにも示されているRCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA等のレクチンである。
　RCA120（RCA I；Ricinus communis Agglutinin I；ヒママメ由来）はGal又はGalNAc特異性が、DSA（Datura stramonium；チョウセンアサガオ由来）はGlcNAc又はポリ-N-アセチルラクトサミン(PolyLacNAc)特異性が、PHA-E4（Phaseolus vulgaris；インゲン豆由来）はGal、GalNAc又はbisecting N-glycan特異性が、SNA（EBL；Elderberry Balk(Sambucus nigra)Lectin；セイヨウニワトコ由来）はSialyl-Gal又はSialyl-GalNAc特異性が、SSA（Sambucus sieboldiana；ニホンニワトコ由来）はシアル酸特異性が、TJA-I（Trichosanthes japonica；キカラスウリ由来）はα2-6結合シアル酸（Neu5Acα2-6Galβ1-3/4GlcNAc）又は6位硫酸化糖鎖（HSO3(-)-6Galβ1-3/4GlcNAc）特異性が、LEL（Lycopersicon esculentum(Tomato)Lectin；トマト由来）は(GlcNAc)n又はポリ-N-アセチルラクトサミン(PolyLacNAc)特異性が、STL(Solanum tuberosum；ジャガイモ由来)は(GlcNAc)n特異性あるいはポリ-N-アセチルラクトサミン(PolyLacNAc)特異性が、そしてConA（Conavalia ensiformis；コンカナバリンA；タチナタマメ由来）は、Man、Glc又はtri-mannosyl N-glycan (core)特異性がそれぞれ知られており、いずれもJ-オイルミルズ社、フナコシ社、コスモ・バイオ株式会社（EY-ラボラトリーズ社）、ベクターラボラトリーズ社などから市販されている。 Typical lectins that can be used as “CSF1R-specific lectins” are RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA, which are also shown in Non-Patent Document 1, Fig. 3B. And other lectins.
RCA120 (RCA I; Ricinus communis Agglutinin I; derived from castor bean) has Gal or GalNAc specificity, DSA (Datura stramonium; derived from datura) has GlcNAc or poly-N-acetyllactosamine (PolyLacNAc) specificity, but PHA-E4 (Phaseolus vulgaris; derived from kidney beans) has Gal, GalNAc or bisecting N-glycan specificity, SNA (EBL; Elderberry Balk (Sambucus nigra) Lectin; derived from elderberry) has Sialyl-Gal or Sialyl-GalNAc specificity, SSA (Sambucus sieboldiana; derived from Japanese elderberry) has sialic acid specificity, while TJA-I (Trichosanthes japonica; derived from Kikarasuuri) has α2-6-linked sialic acid (Neu5Acα2-6Galβ1-3 / 4GlcNAc) or 6-position sulfated sugar chain ( HSO3 (-)-6Galβ1-3 / 4GlcNAc) specificity, LEL (Lycopersicon esculentum (Tomato) Lectin; derived from tomato) (GlcNAc) n or poly-N-acetyllactosamine (PolyLacNAc) specificity, STL (Solanum tuberosum (from potato) is (GlcNAc) n specific or poly-N -Acetyllactosamine (PolyLacNAc) specificity, and ConA (Conavalia ensiformis; Concanavalin A; derived from red bean) are known to have Man, Glc or tri-mannosyl N-glycan (core) specificity, respectively. -Commercially available from Oil Mills, Funakoshi, Cosmo Bio Inc. (EY-Laboratories), Vector Laboratories, etc.
３．本発明において用いる測定方法及びそのためのキット
（３－１）本発明の測定方法及びキットの用途
　本発明において新たに提供された抗CSF1R抗体、並びに抗CSF1R抗体と共にWFA及び／又はVVAレクチンを用いたCSF1R分子、WFA+-CSF1R分子の測定方法及びキットは、本発明における、肝硬変患者の肝細胞がんの発症リスク指数（WFA+-CSF1R％）及び予後判定指数（WFA+-CSF1R ng/ml）の測定用に用いて肝硬変の予後や肝細胞がん発症リスクを判定することができる。
　また、本発明の各キットの構成要素として、WFA／VVA結合性糖鎖含有CSF1R及び／又はWFA／VVA結合性糖鎖非含有CSF1R標準物質をポジコン又はネガコン用に備えることが好ましい。各キットの構成要素は、粉末などの固体状でも、緩衝液などに溶解又は分散した状態の溶液状でもよく、アッセイ用の基板、ビーズなどに結合させた状態でも良い。さらに、溶解又は分散用の緩衝液などをキットの構成要素に加えることもできる。 3. Measurement method and kit for the same used in the present invention (3-1) Use of measurement method and kit of the present invention Anti-CSF1R antibody newly provided in the present invention, and WFA and / or VVA lectin together with anti-CSF1R antibody The method and kit for measuring CSF1R molecule, WFA + -CSF1R molecule are the risk index (WFA + -CSF1R%) and prognosis index (WFA + -CSF1R ng / ml) of hepatocellular carcinoma in cirrhosis patients in the present invention. Can be used to determine the prognosis of cirrhosis and the risk of developing hepatocellular carcinoma.
In addition, as a component of each kit of the present invention, a WFA / VVA-binding sugar chain-containing CSF1R and / or a WFA / VVA-binding sugar chain-free CSF1R standard substance is preferably provided for positive control or negative control. The components of each kit may be in the form of a solid such as a powder, in the form of a solution dissolved or dispersed in a buffer solution, or in a state of being bound to an assay substrate or beads. Furthermore, a buffer for dissolution or dispersion can be added to the components of the kit.
　そして、WFA+-CSF1R分子、すなわちCSF1R上のWFA／VVA結合性糖鎖は、広く各種肝疾患の重篤度を示すマーカーでもあるため（特許文献１）、本発明のWFA+-CSF1R分子測定方法及びキットは、肝疾患の重篤度判定用などに用いることもできる。
　一方、CSF1R分子は、従来から肝疾患マーカー、肝がんマーカーなどとして用いられていたため、本発明のCSF1R分子の測定方法及びキットは、肝疾患診断用又は肝細胞がん診断用などに用いることもできる。
　その際、試料中のCSF1R量は、抗CSF1R抗体によるELISAなどで直接測定することもできるが、CSF1R上のCSF1R特異的レクチン結合性糖鎖量を測定することで、間接的に測定できる。例えば、抗CSF1R抗体と「CSF1R特異的レクチン」とを用いた抗体-レクチンサンドイッチアッセイなどにより試料中の「CSF1R特異的レクチン」反応性糖鎖を有するCSF1R量を測定することで、実質的に試料中の「全CSF1R量」が測定できる。
　体液試料から抗CSF1R抗体アフィニティカラムなど通常のタンパク質精製法を適用してCSF1Rを分離精製し、当該CSF1Rに対するWFA及び又はVVAレクチンの反応量及びCSF1R特異的レクチン反応量を、同時に又は別々に測定し、両測定値の比率を求めることで、肝硬変患者の肝細胞がんの発症リスク指数（WFA+-CSF1R％）を求めることができる。 Since the WFA + -CSF1R molecule, that is, the WFA / VVA-binding sugar chain on CSF1R, is also a marker showing the severity of various liver diseases (Patent Document 1), the WFA + -CSF1R molecule measurement of the present invention is performed. The method and kit can also be used for determining the severity of liver disease.
On the other hand, since CSF1R molecules have been conventionally used as liver disease markers, liver cancer markers, etc., the CSF1R molecule measurement method and kit of the present invention should be used for liver disease diagnosis or hepatocellular carcinoma diagnosis, etc. You can also.
At that time, the amount of CSF1R in the sample can be directly measured by ELISA using an anti-CSF1R antibody, but can be indirectly measured by measuring the amount of CSF1R-specific lectin-binding sugar chain on CSF1R. For example, by measuring the amount of CSF1R having a “CSF1R-specific lectin” reactive sugar chain in the sample by an antibody-lectin sandwich assay using an anti-CSF1R antibody and “CSF1R-specific lectin”, the sample is substantially The "total CSF1R amount" can be measured.
CSF1R is separated and purified from a body fluid sample by applying a conventional protein purification method such as an anti-CSF1R antibody affinity column, and the reaction amount of WFA and / or VVA lectin and the amount of CSF1R-specific lectin reaction against the CSF1R are measured simultaneously or separately. By calculating the ratio of both measured values, the risk index (WFA + -CSF1R%) of hepatocellular carcinoma in patients with cirrhosis can be determined.
（３－２）レクチン－抗体サンドイッチ免疫学的検出法について
　基本的には２種の抗体を用いたサンドイッチ検出法に用いられるプロトコルのうち、一方の抗体をレクチンに置き換えるだけで適用できる。したがって、この手法は既存のELISA法などのほか、自動免疫検出装置を用いた自動化にも適用可能である。唯一考慮しなければならない点は、サンドイッチに用いる抗体とレクチン間の反応である。抗体は、少なくとも２本のN-結合型糖鎖を有する。したがって、使用するレクチンが抗体上の糖鎖を認識する場合は、サンドイッチ検出時にその結合反応に起因するバックグランドノイズを生じてしまう。このノイズシグナルの発生を抑制するのに抗体上の糖鎖部分に修飾を導入する方法や、糖鎖部分を含まないFabのみを用いる方法が考えられるが、これらは公知の手法を用いればよい。糖鎖部分への修飾方法としては、例えばChen SらNat Methods. 4, 437-44 (2007)やComunale MAらJ Proteome Res. 8, 595-602 (2009)等があり、Fabを用いる方法としては例えばMatsumoto HらClin Chem Lab Med 48, 505-512 (2010)等がある。 (3-2) Lectin-antibody sandwich immunological detection method Basically, among the protocols used for the sandwich detection method using two kinds of antibodies, it can be applied only by replacing one antibody with a lectin. Therefore, this method can be applied not only to the existing ELISA method but also to automation using an automatic immunodetection device. The only thing to consider is the reaction between the antibody used in the sandwich and the lectin. The antibody has at least two N-linked sugar chains. Therefore, when the lectin used recognizes a sugar chain on the antibody, background noise due to the binding reaction occurs during sandwich detection. In order to suppress the generation of the noise signal, a method of introducing a modification into the sugar chain part on the antibody or a method of using only a Fab that does not contain a sugar chain part can be considered. Examples of methods for modifying the sugar chain include Chen S et al. Nat Methods. 4, 437-44 (2007) and Comunale MA et al. J Proteome Res. 8, 595-602 (2009). Include, for example, Matsumoto H et al. Clin Chem Lab Med 48, 505-512 (2010).
（３－３）CSF1R上のWFA／VVA結合性結合性糖鎖検出用レクチン－抗体サンドイッチELISA測定系
　WFA／VVA結合性糖鎖を有するCSF1Rのサンドイッチ系での検出には、主としてレクチン‐抗体サンドイッチELISAや、レクチンアレイを用いた抗体オーバーレイ・レクチンアレイ法が用いられる。
　抗CSF1R抗体をELISAプレート固相化側に使用した場合は、レクチンを液相側に検出用として用いる。抗体はELISAプレート固相化側でも検出側（液相側）でも、どちらに使用しても良く、もう一方の側にはレクチンを使用して（つまり抗体が固相側の場合はレクチンを液相側に使用して）、サンドイッチの検出系にて行う。一般的には感度が高く、バックグラウンドとなるノイズが少なくなる組み合わせにて検出系の構築を行う。
　WFA／VVAレクチンとしては、市販のWFAレクチン（天然WFA）、組換えWFA、LDN糖鎖特異的な単量体のsrWFA以外に、VVAレクチンを用いることができる。
　抗CSF1R抗体は、抗CSF1Rモノクローナル抗体を用いることが好ましく、本発明で取得した30クローンのハイブリドーマから採取されるCSR-1～30，好ましくはCSR-3、CSR-4、CSR-18、CSR-21及びCSR-30、又は市販の抗CSF1Rモノクローナル抗体を用いることができる。また、抗CSF1Rモノクローナル抗体の全長を用いる必要はなく、抗原認識部位を有してさえいれば、Fab、F(ab’)2などの抗体フラグメントであってもよく、一本鎖抗体や、bi-specific抗体、あるいは抗原認識部位の配列を人工的に組み換えて他種属抗体化（ヒト化抗体など）した抗体、あるいはその抗体フラグメントでも良い。さらに、抗原に対する結合性を有しているものであれば、ファージディスプレイ抗体（phage display）のようなものでも良い。 (3-3) Lectin-antibody sandwich ELISA system for detection of WFA / VVA-binding sugar chains on CSF1R For detection of CSF1R having WFA / VVA-binding sugar chains in a sandwich system, a lectin-antibody sandwich is mainly used. An ELISA and an antibody overlay / lectin array method using a lectin array are used.
When anti-CSF1R antibody is used on the ELISA plate immobilization side, lectin is used for detection on the liquid phase side. The antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. (Used on the phase side) in a sandwich detection system. In general, a detection system is constructed with a combination of high sensitivity and low background noise.
As the WFA / VVA lectin, VVA lectin can be used in addition to commercially available WFA lectin (natural WFA), recombinant WFA, and LDN sugar chain-specific monomer srWFA.
As the anti-CSF1R antibody, an anti-CSF1R monoclonal antibody is preferably used, and CSR-1 to 30, preferably CSR-3, CSR-4, CSR-18, CSR-, collected from the 30 clone hybridoma obtained in the present invention. 21 and CSR-30, or commercially available anti-CSF1R monoclonal antibodies can be used. Further, it is not necessary to use the full length of the anti-CSF1R monoclonal antibody, and it may be an antibody fragment such as Fab or F (ab ′) 2 as long as it has an antigen recognition site. It may be a -specific antibody, an antibody obtained by artificially recombining the sequence of the antigen recognition site to produce an antibody of another species (such as a humanized antibody), or an antibody fragment thereof. Furthermore, a substance such as a phage display antibody (phage display) may be used as long as it has a binding property to an antigen.
（３－４）レクチン又は抗体の標識法
　マーカー分子であるCSF1R又はCSF1R上の糖鎖を検出する際、レクチンと抗体の組み合わせによるサンドイッチELISA測定系の感度を上げるため、化学発光を用いた検出系（化学発光酵素免疫測定法、Chemiluminescent Enzyme Immunoassay；CLEIA法）を適用することもできる。
　サンドイッチアッセイをするために使用する二次抗体として、検出側が抗体の場合は、西洋ワサビペルオキシダーゼ（HRP）などでラベル化した抗マウスIgG抗体などで検出し、発色させることが一般的であるが、この一次抗体としてビオチン標識された抗体を使用したビオチン－アビジン反応を利用する検出系を利用することも出来る。一方、検出側がWFAレクチン、VVAレクチンなどレクチンの場合、レクチン検出用二次抗体を用いるよりも、ビオチン標識されたレクチンを使用したビオチン－アビジン反応を利用する検出系が簡便であり好ましい。
　具体的には、ビオチン標識化したWFAレクチン、VVAレクチンなどを反応させた後、溶液を廃棄して洗浄後、HRP標識ストレプトアビジン溶液を反応させ、反応液を廃棄、洗浄後、TMB基質液による発色を観察すればよい。
また、上記検出用の抗体やレクチンについて、ビオチン標識の代わりに蛍光物質による標識を行うことで、抗体やレクチンが結合していることを直接検出する系（化学発光に依らない系）を構築することも出来る。 (3-4) Labeling method of lectin or antibody A detection system using chemiluminescence is used to increase the sensitivity of sandwich ELISA measurement system by combination of lectin and antibody when detecting sugar chain on marker molecule CSF1R or CSF1R (Chemiluminescent enzyme immunoassay; Chemiluminescent Enzyme Immunoassay; CLEIA method) can also be applied.
As a secondary antibody used for sandwich assay, when the detection side is an antibody, it is common to detect and develop color with an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) etc. A detection system using a biotin-avidin reaction using a biotin-labeled antibody as the primary antibody can also be used. On the other hand, when the detection side is a lectin such as a WFA lectin or a VVA lectin, a detection system using a biotin-avidin reaction using a biotin-labeled lectin is simpler and preferable than using a secondary antibody for lectin detection.
Specifically, after reacting biotin-labeled WFA lectin, VVA lectin, etc., discard the solution, wash, react with HRP-labeled streptavidin solution, discard the reaction solution, wash, and then use the TMB substrate solution What is necessary is just to observe color development.
In addition, the detection antibody or lectin is labeled with a fluorescent substance instead of a biotin label, thereby constructing a system (system that does not depend on chemiluminescence) that directly detects the binding of the antibody or lectin. You can also
（３－５）試料中のCSF1Rの検出系
　体液（血清）試料中の全CSF1R量を測定するためには、既知のウェスタンブロッティング法、ELISA法、サンドイッチELISA法、が使用可能であり、他にレクチンアレイを用いた抗体オーバーレイ・レクチンアレイ法、定量的な質量分析法（LC-MS等）、免疫学的測定法、酵素活性測定法、キャピラリー電気泳動法、液体クロマトグラフィー（HPLC）法、薄層クロマトグラフィー法、金コロイド法、放射免疫測定法、ラテックス凝集免疫測定法、蛍光免疫測定法、ウェスタンブロッティング法、免疫組織化学法、表面プラズモン共鳴法（SPR法）又は水晶振動子マイクロバランス（QCM）法等の分離・検出方法の他、マイクロフリュイディクス技術を用いた分離・検出システムが適用できる。抗体－抗体サンドイッチELISA法が好ましい。
　その際の抗CSF1R抗体としては、（３－３）で述べた抗CSF1Rモノクローナル抗体を用いることが好ましく、二次抗体についても同様である。 (3-5) Detection System for CSF1R in Samples To measure the total amount of CSF1R in body fluid (serum) samples, known Western blotting, ELISA, and sandwich ELISA methods can be used. Antibody overlay / lectin array method using lectin array, quantitative mass spectrometry (LC-MS, etc.), immunological assay, enzyme activity assay, capillary electrophoresis, liquid chromatography (HPLC), thin Layer chromatography method, gold colloid method, radioimmunoassay method, latex agglutination immunoassay method, fluorescence immunoassay method, western blotting method, immunohistochemistry method, surface plasmon resonance method (SPR method) or quartz crystal microbalance (QCM) In addition to separation / detection methods such as), separation / detection systems using microfluidics technology can be applied. The antibody-antibody sandwich ELISA method is preferred.
As the anti-CSF1R antibody at that time, the anti-CSF1R monoclonal antibody described in (3-3) is preferably used, and the same applies to the secondary antibody.
（３－６）全CSF1R量に対応する「CSF1R特異的レクチン結合性糖鎖含有CSF1R量」の検出系
　WFA+-CSF1R％を算出するための全CSF1R量に代わる「CSF1R特異的レクチン結合性糖鎖含有CSF1R量」、すなわち体液（血清）中のCSF1R上の「CSF1R特異的レクチン結合性糖鎖」の量は、「CSF1R特異的レクチン」と抗CSF1R抗体を用い、（３－２）で述べたレクチン－抗体サンドイッチELISA測定系により測定した値を用いることができる。その他の検出方法についても、上記と同じような分離・検出方法が適用できる。
　ここで、「CSF1R特異的レクチン」としては、RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA等が候補になる。
　例えば「全CSF1R量」を測定するための2種類の抗CSF1R抗体を用いる抗体-抗体サンドイッチアッセイに代えて、RCA120（及び／又はDSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA）と抗CSF1R抗体とを用いるレクチン-抗体サンドイッチアッセイを用いることができる。WFA（及び／又はVVA）と共にRCA120（及び／又はDSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA）を含むレクチンアレイを用い、体液試料、好ましくは予め抗CSF1R抗体抗CSF1R抗体をオーバーレイすれば一度の測定で「WFA+-CSF1R％」を求めることができるといえる。 (3-6) "CSF1R-specific lectin-binding saccharide-containing CSF1R-specific lectin-binding saccharide" in place of the total CSF1R amount for calculating "CSF1R-specific lectin-binding sugar chain-containing CSF1R amount" detection system WFA + -CSF1R% The amount of “chain-containing CSF1R”, that is, the amount of “CSF1R-specific lectin-binding sugar chain” on CSF1R in body fluid (serum) is described in (3-2) using “CSF1R-specific lectin” and anti-CSF1R antibody. The value measured by the lectin-antibody sandwich ELISA measurement system can be used. As for other detection methods, the same separation / detection method as described above can be applied.
Here, as the “CSF1R-specific lectin”, RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA and the like are candidates.
For example, instead of an antibody-antibody sandwich assay using two types of anti-CSF1R antibodies to measure “total CSF1R amount”, RCA120 (and / or DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL) , ConA) and anti-CSF1R antibodies can be used for lectin-antibody sandwich assays. Using a lectin array containing RCA120 (and / or DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA) together with WFA (and / or VVA), body fluid samples, preferably anti-CSF1R antibody If the CSF1R antibody is overlaid, it can be said that “WFA + -CSF1R%” can be obtained in one measurement.
（３－５）レクチンと抗CSF1R抗体とを用いたその他の検出系について
　前記（３－２）で述べたように、試料中のWFA及び／又はVVA（WFA／VVA）レクチンと特異的に結合する糖鎖を有するCSF1Rは、レクチン‐抗体サンドイッチELISAや、レクチンアレイを用いた抗体オーバーレイ・レクチンアレイ法で簡便に検出又は定量が可能であるが、他の具体的な方法として、例えば、以下の方法が挙げられる。
（１）被験者から得られた体液（血清）中のCSF1R分子を、WFA及び／又はVVAレクチンを用いて分離する。これによって、WFA／VVAレクチンと特異的に結合する糖鎖を有するタンパク質群が選択される。
（２）続いて、WFA／VVAレクチンと特異的に結合する糖鎖以外の部分を特異的に認識する抗CSF1R抗体を用いて検出する。これによって、目的とするWFA／VVAレクチンと特異的に結合する糖鎖を有するCSF1Rマーカーを検出することができる。
　WFA／VVAレクチンと特異的に反応する糖鎖を有するCSF1Rの検出には、例えば、WFA／VVAレクチンに特異的に結合するCSF1Rを測定する方法、具体的には、WFA／VVAレクチンを固定したカラムやアレイによる捕集や分離を行う方法、及びCSF1Rを測定する手段、具体的には、CSF1R糖タンパク質の質量分析などによる直接検出又はそのCSF1R（断片含む）に対する抗体を用いて行なうことができる。先にレクチンと抗体のサンドイッチによる複合体形成を行ってから、後述の方法で分離し、これを検出することもできる。ウェスタンブロット法によってもWFA／VVA結合性糖鎖を有するCSF1R糖タンパク質（糖ペプチド）を検出、定量することができる。 (3-5) Other detection system using lectin and anti-CSF1R antibody As described in (3-2) above, it specifically binds to WFA and / or VVA (WFA / VVA) lectin in the sample. CSF1R having a sugar chain that can be easily detected or quantified by lectin-antibody sandwich ELISA or antibody overlay lectin array method using lectin array, but other specific methods include, for example, A method is mentioned.
(1) CSF1R molecules in body fluid (serum) obtained from a subject are separated using WFA and / or VVA lectin. Thus, a protein group having a sugar chain that specifically binds to the WFA / VVA lectin is selected.
(2) Subsequently, detection is performed using an anti-CSF1R antibody that specifically recognizes a portion other than the sugar chain that specifically binds to the WFA / VVA lectin. Thereby, the CSF1R marker having a sugar chain that specifically binds to the target WFA / VVA lectin can be detected.
For detection of CSF1R having a sugar chain that specifically reacts with WFA / VVA lectin, for example, a method of measuring CSF1R specifically binding to WFA / VVA lectin, specifically, fixing WFA / VVA lectin A method for collecting and separating by a column or array, and a means for measuring CSF1R, specifically, direct detection by mass spectrometry of CSF1R glycoprotein or using an antibody against the CSF1R (including fragment) . A complex formed by a lectin-antibody sandwich can be formed first, and then separated by the method described later to detect this. The CSF1R glycoprotein (glycopeptide) having a WFA / VVA-binding sugar chain can also be detected and quantified by Western blotting.
　反対に、予め分離精製しておいたCSF1R糖タンパク質をレクチンアレイなどのチップ・装置を用いて、固相化された複数のレクチンに対するCSF1R糖タンパク質の結合量（マルチレクチンアッセイ）を検出することでも、WFA／VVA結合性糖鎖を有するCSF1R分子の測定を行うことが出来る。例えば、以下の手順で行う。
（１）被験者から得られた体液（血清）試料中より、CSF1R（断片含む）に対する抗体を使用してCSF1R糖タンパク質を分離精製する。
（２）続いて、分離精製したCSF1R糖タンパク質をレクチンアレイに供し、蛍光標識された抗CSF1R抗体を添加して複合体を形成させ、アレイスキャナーにて各レクチンスポットの蛍光強度（つまりCSF1R糖タンパク質の各レクチンへの結合量）を測定する。これによって、目的とするWFA／VVAレクチンと特異的に結合する糖鎖を有するCSF1Rマーカー、およびCSF1R特異的レクチンに結合するCSF1Rマーカー、全CSF1Rマーカーを測定することができる。
　また、（１）で得られたCSF1R糖タンパク質は、WFA／VVAレクチンを使用したレクチンブロット法によっても検出、定量することができる。 On the other hand, it is also possible to detect the amount of CSF1R glycoprotein bound to multiple immobilized lectins (multilectin assay) using a lectin array or other chip / equipment of CSF1R glycoprotein that has been separated and purified in advance. , CSF1R molecules having WFA / VVA-binding sugar chains can be measured. For example, the following procedure is used.
(1) CSF1R glycoprotein is separated and purified from a body fluid (serum) sample obtained from a subject using an antibody against CSF1R (including fragments).
(2) Subsequently, the separated and purified CSF1R glycoprotein is applied to a lectin array, a fluorescently labeled anti-CSF1R antibody is added to form a complex, and the fluorescence intensity (that is, CSF1R glycoprotein of each lectin spot) is detected by an array scanner. The amount of binding to each lectin) is measured. Thereby, the CSF1R marker having a sugar chain that specifically binds to the target WFA / VVA lectin, the CSF1R marker that binds to the CSF1R-specific lectin, and all CSF1R markers can be measured.
The CSF1R glycoprotein obtained in (1) can also be detected and quantified by a lectin blot method using WFA / VVA lectin.
（３－７）その他の検出系
　その他の検出系としては、定量的な質量分析法（LC-MS等）、免疫学的測定法、酵素活性測定法、キャピラリー電気泳動法、液体クロマトグラフィー（HPLC）法、薄層クロマトグラフィー等が挙げられる。好適には、LC-MS、WFA（VVA）レクチンと特異的に反応する糖鎖を有するCSF1R糖タンパク質又はその断片に特異的なモノクローナル抗体又はポリクローナル抗体を用いた、酵素免疫測定法、二抗体サンドイッチELISA法、金コロイド法、放射免疫測定法、ラテックス凝集免疫測定法、蛍光免疫測定法、ウェスタンブロッティング法、免疫組織化学法、表面プラズモン共鳴法（SPR法）又は水晶振動子マイクロバランス（QCM）法等による定性的又は定量的手法を用いることができる。 (3-7) Other detection systems Other detection systems include quantitative mass spectrometry (LC-MS, etc.), immunoassay, enzyme activity measurement, capillary electrophoresis, liquid chromatography (HPLC ) Method, thin layer chromatography and the like. Preferably, LC-MS, enzyme immunoassay using a monoclonal antibody or polyclonal antibody specific for CSF1R glycoprotein having a sugar chain that specifically reacts with WFA (VVA) lectin or a fragment thereof, two-antibody sandwich ELISA method, gold colloid method, radioimmunoassay method, latex agglutination immunoassay method, fluorescence immunoassay method, western blotting method, immunohistochemistry method, surface plasmon resonance method (SPR method) or quartz crystal microbalance (QCM) method Qualitative or quantitative techniques such as can be used.
　質量分析による検出方法としては以下の通りに実施することができる。マーカー糖ペプチド及び糖タンパク質は、糖鎖に結合するプローブレクチン、あるいは作製された抗CSF1R抗体で捕集した試料について、質量分析計を検出器として使用し、LacdiNAc（LDN）糖鎖を有する糖ペプチドを検出することができる。マーカー糖ペプチドの検出は、好適には捕集した糖ペプチドの糖鎖を切除処理した後、液体クロマトグラフィー（LC）で分離し、溶出したペプチドを順次、直接オンラインで質量分析計（MS）に導入し、検出することができる。質量スペクトルの収集は、単に質量スペクトルを取得することに加え、衝突誘起解離（CID）等の破断法を利用したMS/MSスペクトルの取得、さらには、予め選択したイオンが検出された場合のみCID等で破断し、生じた複数のフラグメントイオンを検出すること（シングルリアクションモニタリング、あるいはマルチリアクションモニタリングと呼ばれる手法）もできる。さらに分析試料に、肝臓がんマーカー糖ペプチドのコアペプチド部分を合成し、その一部に安定同位体を取り込ませて質量差を生じさせた対象ペプチドを加え、それぞれのシグナル強度を比較することで相対的あるいは絶対的定量分析を行うこともできる。簡易的には検出されたイオンのシグナル強度を複数の試料間あるいは標準試料と比較し、簡易定量することもできる。 The detection method by mass spectrometry can be carried out as follows. Marker glycopeptides and glycoproteins are glycopeptides that have LacdiNAc (LDN) sugar chains using a mass spectrometer as a detector for samples collected with the probe lectin that binds to the sugar chain or the prepared anti-CSF1R antibody. Can be detected. The marker glycopeptide is preferably detected by excising the sugar chain of the collected glycopeptide, separating it by liquid chromatography (LC), and then eluting the peptide directly and directly on-line to a mass spectrometer (MS). Can be introduced and detected. In addition to acquiring mass spectra, mass spectra can be acquired by acquiring MS / MS spectra using a fracture method such as collision-induced dissociation (CID), and only when preselected ions are detected. It is also possible to detect a plurality of fragment ions that are broken by a method such as single reaction monitoring or multi-reaction monitoring. Furthermore, by synthesizing the core peptide part of the liver cancer marker glycopeptide to the analysis sample, adding the target peptide that has incorporated a stable isotope into a part of it and causing a mass difference, and comparing the signal intensity of each Relative or absolute quantitative analysis can also be performed. In a simple manner, the signal intensity of the detected ions can be easily quantified by comparing between a plurality of samples or a standard sample.
　また、臨床検体試料から抗CSF1R抗体により分離精製したCSF1Rに対し、WFA（VVA）レクチンを作用させてキャピラリー電気泳動法（Kuroda Y.,et al.,Pharm Res.,2001 Mar;18(3):389-93）や、マイクロフリュイディクス技術（横山ら「生物試料分析」Vol.33,No.3(2010)p.201-206）に適用することで、分子量の違いとしてWFA／VVAレクチン反応性CSF1Rタンパク質と、非反応性CSF1Rタンパク質との量比を測定し、全CSF1R中のWFA／VVA結合性CSF1Rの量が算出できる。また、被検体液試料由来精製CSF1Rに対して、WFA／VVAレクチン及びRCA120（又はDSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA）レクチンをそれぞれ作用させ、２種類のレクチン-CSF1Rタンパク質複合体を形成させて、マイクロフリュイディクス技術を適用することによっても、各レクチンの分子量の差に基づき、被検体液試料中のWFA／VVAレクチンと結合したCSF1R量及びCSF1R特異的レクチンと結合したCSF1R量がそれぞれ測定できる。そして、両者の量比を求めることで、全CSF1R量に対するWFA／VVA結合性糖鎖含有CSF1Rの量比が決定できる。具体的な測定用装置としては、キャピラリー電気泳動装置や、μTASWako i30（和光純薬工業（社））のような、マイクロフリュイディクス技術・分離／検出技術を用いた装置などを使用することができる。 In addition, capillary electrophoresis (Kuroda Y., et al., Pharm Res., 2001 Mar; 18 (3)) was applied to WSF (VVA) lectin on CSF1R isolated and purified from anti-CSF1R antibodies from clinical specimens. : 389-93) and microfluidics technology (Yokoyama et al. “Biological Sample Analysis” Vol.33, No.3 (2010) p.201-206) By measuring the quantitative ratio between the sex CSF1R protein and the non-reactive CSF1R protein, the amount of WFA / VVA-binding CSF1R in the total CSF1R can be calculated. In addition, WFA / VVA lectin and RCA120 (or DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA) lectin are allowed to act on the purified CSF1R derived from the sample liquid sample, respectively. Based on the difference in the molecular weight of each lectin by forming a lectin-CSF1R protein complex and applying microfluidics technology, the amount of CSF1R bound to WFA / VVA lectin in the sample fluid sample and CSF1R specific The amount of CSF1R bound to lectin can be measured respectively. And by calculating | requiring the amount ratio of both, the amount ratio of WFA / VVA binding sugar chain containing CSF1R with respect to the total amount of CSF1R can be determined. As a specific measurement apparatus, a capillary electrophoresis apparatus, an apparatus using microfluidics technology / separation / detection technology, such as μTASWako i30 (Wako Pure Chemical Industries, Ltd.), and the like can be used. .
　以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。
　本発明におけるその他の用語や概念は、当該分野において慣用的に使用される用語の意味に基づくものであり、本発明を実施するために使用する技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。また、各種の分析などは、使用した分析機器又は試薬、キットの取り扱い説明書、カタログなどに記載の方法を準用して行った。
　なお、本明細書中に引用した技術文献、特許公報及び特許出願明細書中の記載内容は、本発明の記載内容として参照されるものとする。 EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and the techniques used to implement the present invention are not specifically limited to the techniques that clearly indicate the source. Can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.
　本実施例で用いた臨床試験方法、測定法、解析方法を以下説明する。
（臨床検査）
　本実施例で用いる血清試料は、試験に使用されるまで-80℃で凍結保存され用事解凍された。本実施例で行われた臨床検査は、血小板数、プロトロンビン活性時間（PT）、血清中アスパラギン酸アミノトランスフェラーゼ濃度（AST）、血清アラニン・アミノトランスフェラーゼ濃度（ALT）、血清アルブミン、血清中総ビリルビン（T.bil）について常法で行った。血清中αフェトプロテイン（AFP）、AFP-LCAレクチン分画（AFP-L3、％）、ビタミンK依存性凝固因子前駆体II（PIVKA-II）も初診時に同じ試料で測定をした。血清AFPはHISCL-2000i（シスメックス）、PIVKA-IIはルミパルスPrestoII（富士レビオ）自動化学発光酵素免疫測定装置（CLEIA）を用いて測定した。従来のAFP-L3は自動免疫測定装置　μTASWako i30（和光純薬）を用いてレクチン親和クロマトグラフィーおよび液相結合アッセイ法で測定した。 The clinical test method, measurement method, and analysis method used in this example will be described below.
(Clinical examination)
The serum samples used in this example were stored frozen at −80 ° C. and thawed until use for testing. The clinical tests performed in this example were platelet count, prothrombin activation time (PT), serum aspartate aminotransferase concentration (AST), serum alanine aminotransferase concentration (ALT), serum albumin, serum total bilirubin ( T.bil) was conducted in a conventional manner. Serum alpha-fetoprotein (AFP), AFP-LCA lectin fraction (AFP-L3,%), and vitamin K-dependent coagulation factor precursor II (PIVKA-II) were also measured in the same sample at the first visit. Serum AFP was measured using HISCL-2000i (Sysmex), and PIVKA-II was measured using a Lumipulse Presto II (Fujirebio) automated chemiluminescent enzyme immunoassay device (CLEIA). Conventional AFP-L3 was measured by lectin affinity chromatography and liquid phase binding assay using an automated immunoassay device μTASWako i30 (Wako Pure Chemical Industries).
（抗CSF1R抗体-WFAレクチン サンドイッチELISAおよび総CSF1R-ELISA）
　WFA+-CSF1Rおよび総CSF1Rは、本発明者らの既報（非特許文献２）の方法を一部改変して行った。Maxisorp（登録商標）イムノプレート（サーモサイエンティフックNUNC、449824）を4μg/mL抗CSF1R抗体（マウス抗ヒトM-CSFR、MAB3292、R&D Systems社）で6時間コートし、ブロッキングバッファー（3％ BSA含有PBSバッファー, pH 7.4）で終夜4℃にてブロッキングした。血清試料はブロッキングバッファーで20倍希釈し、2対をプレートに2時間アプライした。次にプレートをウォッシュバッファーで6回洗浄した。ビオチン結合WFA（ベクターラボラトリ社）が検出プローブとして用いられた。プレートは100μL/well の50,000倍希釈HRP結合ストレプトアビジン溶液とともにインキュベートしウォッシュバッファーで6回洗浄した。各ウェルに基質溶液（100μL、サーモフィッシャーサイエンティフィック）を加え、反応は1M H2SO4で止めた。各ウェルの450nmにおける吸光度を測定した。 (Anti-CSF1R antibody-WFA lectin sandwich ELISA and total CSF1R-ELISA)
WFA + -CSF1R and total CSF1R were obtained by partially modifying the method of the present inventors' report (Non-patent Document 2). Maxisorp® immunoplate (Thermo Scientific Hook NUNC, 449824) was coated with 4 μg / mL anti-CSF1R antibody (mouse anti-human M-CSFR, MAB3292, R & D Systems) for 6 hours, and blocking buffer (containing 3% BSA) Blocking was continued overnight at 4 ° C with PBS buffer, pH 7.4). Serum samples were diluted 20-fold with blocking buffer and 2 pairs were applied to the plate for 2 hours. The plate was then washed 6 times with wash buffer. Biotin-conjugated WFA (Vector Laboratories) was used as a detection probe. The plate was incubated with 100 μL / well of a 50,000-fold diluted HRP-conjugated streptavidin solution and washed 6 times with wash buffer. Substrate solution (100 μL, Thermo Fisher Scientific) was added to each well and the reaction was stopped with 1M H 2 SO 4 . The absorbance at 450 nm of each well was measured.
（観察期間およびHCCの治療）
　患者の経過観察は腫瘍マーカーAFP、PIVKA-II、AFP-L3、および超音波検査、CT、磁気共鳴画像法を用いて少なくとも開始より6か月ごとに行った。HCC治療後最初の一年間の経過観察は3か月ごとに画像診断で行われ、この期間中、肺炎、敗血症、HCCを含む肝臓に関連する死亡、および食道静脈瘤出血を含む肝不全による死亡について分析した。
　HCCは日本のガイドラインに従って治療された。まず患者は手術の適応について評価され、外科的治療の拒否もしくは不適格とされた場合、経皮的エタノール注入法による局所凝固療法（LAT）、または近年では高周波アブレーション（RFA）が行われた。肝移植を受けた患者はいなかった。各HCC患者のフォローアップ期間は1998年から2014年のうちに開始され患者の死亡もしくは2014年8月まで続けられた。フォローアップ期間は1か月から195ヶ月（中央値60ヶ月）だった。 (Observation period and treatment of HCC)
Patient follow-up was performed at least every 6 months from the start using tumor markers AFP, PIVKA-II, AFP-L3, and ultrasonography, CT, and magnetic resonance imaging. The first year of follow-up after HCC treatment is imaged every 3 months, during which time pneumonia, sepsis, liver-related deaths including HCC, and deaths due to liver failure including esophageal varices bleeding Was analyzed.
HCC was treated according to Japanese guidelines. Patients were first evaluated for surgical indications, and if they refused or were ineligible for surgical treatment, local coagulation (LAT) with percutaneous ethanol injection, or more recently radiofrequency ablation (RFA), was performed. None of the patients received a liver transplant. The follow-up period for each HCC patient started between 1998 and 2014 and continued until patient death or August 2014. The follow-up period ranged from 1 to 195 months (median 60 months).
（統計学的解析）
　本実施例で用いる統計解析は、患者の臨床背景はMann-WhitneyのU検定を使用、累積生存率および発がん率はカプラン・マイヤー（Kaplan-Meier）法で算出，検定はLog rank testで行った。WFA+-CSF1R、WFA+-CSF1R％は最小P値法を用いて最適なカットオフ値を算出した。変数の最適なカットオフ値を決定するため、ログラング検定の10から90パーセンタイル順位間で最小のP値を示すカットオフ値を選択した。危険率（HR）と95％信頼区間（95％CI）も求められた。生存率および累積発がんの予測因子は、年齢、性別、アルブミン値、血小板数、Fib4、APRI、AFP、PIVKA-II、およびAFP-L3を検討した。時間依存性ROC解析にてWFA+-CSF1R、WFA+-CSF1R％の有用性を検討した。連続型変数間の相関はスピアマンの順位相関係数で数量化された。また、P＜0.05を統計的有意とした。統計学的分析はSPSS.20, R 2.14.0 （survival ROCパッケージ）およびWindows Excel 2010などの統計解析用ソフトウェアを用いて行った。 (Statistical analysis)
In the statistical analysis used in this example, Mann-Whitney U test was used for clinical background of patients, cumulative survival rate and carcinogenic rate were calculated by Kaplan-Meier method, and the test was performed by Log rank test . For WFA + -CSF1R and WFA + -CSF1R%, the optimal cutoff value was calculated using the minimum P-value method. To determine the optimal cut-off value for the variable, the cut-off value showing the smallest P value between the 10th and 90th percentile ranks of the log Lang test was selected. A risk factor (HR) and 95% confidence interval (95% CI) were also determined. Predictors of survival and cumulative carcinogenesis were examined for age, gender, albumin level, platelet count, Fib4, APRI, AFP, PIVKA-II, and AFP-L3. The usefulness of WFA + -CSF1R and WFA + -CSF1R% was examined by time-dependent ROC analysis. The correlation between continuous variables was quantified by Spearman's rank correlation coefficient. P <0.05 was considered statistically significant. Statistical analysis was performed using statistical analysis software such as SPSS.20, R 2.14.0 (survival ROC package) and Windows Excel 2010.
（実施例１）免疫組織化学染色法によるCSF1R糖タンパク質の解析
　各種肝疾患、特に肝硬変あるいは肝臓がんの患者の組織から薄切された標本（凍結標本あるいはパラフィン包埋標本）を使用して、レクチン或いは抗体による免疫組織化学的染色による発現の検討を行うことができる。そこで、組織アレイスライド（ホルマリン固定パラフィン包埋ブロックより作製されたもの）を使用して、肝臓がん組織におけるWFAならびにCSF1R発現の検討を行った。
　組織アレイスライドを脱パラフィンした後、蒸留水にて洗浄し、100 mM クエン酸バッファー（pH 9.0）中で電子レンジ（microwave oven）にて5分間加熱して抗原賦活化を行った。次いで、内因性peroxidaseの阻害処理を行い、ブロッキングバッファー (0.2％ Tween-20, 5％ glycerol, 3％ BSA in PBS)にて、室温で20分ブロッキングを行った。PBS中で3回洗浄した後、ブロッキングバッファーで希釈した1次抗体（抗CSF1R抗体：C20クローン、Santa Cruz Biotechnology社, 1 μg/mLにて使用。またはR&D systems社AF329、抗hMCSFR抗体）あるいは、ビオチン化WFAレクチン：Vector Laboratories社, 20μg/mLにて使用）にて反応させた。一次反応後、PBS中で3回洗浄し、次に2次抗体として、2次抗体（HRP化抗ウサギIgG抗体： Life Technologies社, 10μg/mLにて使用）あるいは、HRPラベル化ストレプトアビジン：Vector Laboratories社, 20μg/mLにて使用）にて反応させた。場合によってVECTASTAIN ABC ELITEキットを使用することも出来る。発色はDAB発色（WAKO社）にて行った。また、haematoxylinにて染色を行った。
その結果、図１に示す通り、肝細胞がん患者組織において、がん組織の辺縁部（浸潤部）にCSF1R分子ならびにWFAエピトープの共発現を認めた。また、図２に示す通り、肝細胞がん患者組織アレイにおいて、CSF1R分子の発現を認めたものは78例/100例中であり、WFAエピトープの発現を認めたものは76例/100例中であった。CSF1R分子ならびにWFAエピトープの共発現を認めたものは、70例/100例中であった。 (Example 1) Analysis of CSF1R glycoprotein by immunohistochemical staining method Using specimens (frozen specimens or paraffin-embedded specimens) sliced from tissues of patients with various liver diseases, particularly cirrhosis or liver cancer, Expression by immunohistochemical staining with lectin or antibody can be examined. Therefore, we examined the expression of WFA and CSF1R in liver cancer tissues using tissue array slides (made from formalin-fixed paraffin-embedded blocks).
The tissue array slide was deparaffinized, washed with distilled water, and antigen-activated by heating for 5 minutes in a microwave oven (microwave oven) in 100 mM citrate buffer (pH 9.0). Next, endogenous peroxidase inhibition treatment was performed, and blocking was performed with a blocking buffer (0.2% Tween-20, 5% glycerol, 3% BSA in PBS) at room temperature for 20 minutes. After washing 3 times in PBS, diluted with blocking buffer (anti-CSF1R antibody: C20 clone, used in Santa Cruz Biotechnology, 1 μg / mL. Or R & D systems AF329, anti-hMCSFR antibody) or Biotinylated WFA lectin: Vector Laboratories, used at 20 μg / mL). After the primary reaction, it was washed 3 times in PBS, and then secondary antibody (HRP anti-rabbit IgG antibody: used at Life Technologies, 10 μg / mL) or HRP-labeled streptavidin: Vector Laboratories, used at 20 μg / mL). In some cases, the VECTASTAIN ABC ELITE kit can be used. Coloring was performed with DAB coloring (WAKO). Moreover, it dye | stained with haematoxylin.
As a result, as shown in FIG. 1, co-expression of CSF1R molecule and WFA epitope was recognized in the peripheral part (infiltrating part) of the cancer tissue in the tissue of the hepatocellular carcinoma patient. In addition, as shown in Fig. 2, in the tissue array of hepatocellular carcinoma patients, CSF1R molecule expression was observed in 78 cases / 100 cases, and WFA epitope expression was observed in 76 cases / 100 cases. Met. In 70/100 cases, co-expression of CSF1R molecule and WFA epitope was observed.
（実施例２）肝硬変（LC）に罹患し、かつ肝細胞がん（HCC）に罹患していない臨床試験患者（LC(+)HCC(-)）の選択
　1998年1月から2013年1月にかけて、名古屋市立大学病院通院中のC型慢性肝疾患患者214名から得られた血清を使用した。HBs抗原陽性患者、およびエントリー時から3か月以内に他臓器の悪性疾患を発症した患者は除外した。観察期間中央値は60ヶ月間（1～195ヶ月間）で、チャイルド・ピュー分類Cの患者は、転院などにより発がん率や予後が正確に評価できないため検討から除外した。肝線維化評価は、肝生検組織または超音波やCTによりおこなった。肝細胞がんは組織学的検査また画像診断により、米国肝臓病学会の基準に基づき診断した。線維化ステージの評価はMETAVIRを用いて2名の病理専門医が個別に判定し、肝硬変をF4とした。本研究は1975年のヘルシンキ宣言をもとに、名古屋市立大学病院倫理委員会で承認され，書面上で同意を得た上で実施した。
　患者選択を図３に示す。214名（慢性肝炎［CH］99名、肝硬変［LC］115名）がエントリーされ、LC115名のうち、肝細胞がん合併は59名（HCC-LC）であった。最終的に、肝がんコントロール不良（23名）もしくは3cm・3個以上の肝細胞がんを有する27名が除外された。さらに長崎医療センター、新松戸中央総合病院、および久留米大学から得られた45名の肝細胞がん合併のない肝硬変患者検体をバリデーションコホートとして検討した。バリデーションコホートは年齢中央値62歳、男性20名（44.4％）観察期間中央値は60ヶ月であった（1～180ヶ月）。これら2グループに年齢とALT値以外の基準特性に著しい違いはなかった（表１）。 (Example 2) Selection of clinical trial patients (LC (+) HCC (-)) suffering from cirrhosis (LC) and not suffering from hepatocellular carcinoma (HCC) from January 1998 to January 2013 In the meantime, sera obtained from 214 patients with chronic liver disease type C who visited Nagoya City University Hospital were used. HBs antigen positive patients and patients who developed malignant diseases of other organs within 3 months from the time of entry were excluded. The median observation period was 60 months (1 to 195 months), and patients with Child Pew classification C were excluded from the study because the cancer rate and prognosis could not be accurately assessed due to transfer. Liver fibrosis was evaluated by liver biopsy tissue, ultrasound, or CT. Hepatocellular carcinoma was diagnosed by histological examination or diagnostic imaging based on the criteria of the American Society of Liver Disease. The fibrosis stage was evaluated individually by two pathologists using METAVIR, and cirrhosis was defined as F4. This study was approved by the Nagoya City University Hospital Ethics Committee based on the 1975 Declaration of Helsinki, with written consent.
Patient selection is shown in FIG. 214 patients (chronic hepatitis [CH] 99 patients, cirrhosis [LC] 115 patients) were entered, and among the 115 LC patients, 59 (HCC-LC) had hepatocellular carcinoma. Eventually, 27 patients with poor liver cancer control (23 patients) or 3 cm · 3 or more hepatocellular carcinomas were excluded. In addition, 45 cirrhosis patients without hepatocellular carcinoma from Nagasaki Medical Center, Shin-Matsudo Central General Hospital, and Kurume University were examined as a validation cohort. The validation cohort had a median age of 62 years and 20 men (44.4%). The median observation period was 60 months (1 to 180 months). There were no significant differences in criteria characteristics other than age and ALT values in these two groups (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　そこで、以下の実施例では、これら2つのコホートを合わせて解析することとし、特にエントリー時に肝細胞がんを有さない101名のLC患者（LC＋HCC－）において生存率と累積発がん率を検討した（表２）。 In the following examples, the two combined cohorts and to analyze, especially at the entry LC patients 101 patients without hepatocellular carcinoma - cumulative carcinogenic and survival in (LC + HCC) (Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（実施例３）肝硬変患者（LC）の肝細胞がん発症リスクの予測のための指標
　本実施例では、肝硬変患者（LC）のうちで肝細胞がん発症リスクを予測のための指標の検討を行う。 (Example 3) Indicators for predicting the risk of developing hepatocellular carcinoma in patients with cirrhosis (LC) In this example, examination of indicators for predicting the risk of developing hepatocellular carcinoma among patients with cirrhosis (LC) I do.
（３－１）全患者血清中WFA +-CSF1R 濃度およびWFA+-CSF1R％
　肝疾患患者全体（214名）の血清試料を用いてWFA+-CSF1Rおよび総CSF1R濃度を測定したところ、血清WFA +-CSF1R 濃度、総CSF1R濃度ともに、肝硬変患者（LC）（115名）は肝炎患者（CH）（99名）に比べ有意に高値だった[WFA +-CSF1R：208.9 (34.3 - 500.9) ng/ml vs. 82.3 (5.0 - 241.0) ng/ml] 、[総CSF1R：845.3 (431.6 - 1487.5) ng/ml vs. 536.4 (266.3 - 1357.2) ng/ml]。
　LC患者115名（HCC 59名、非HCC 56名）において血清WFA +-CSF1R濃度 [208.9 (85.4 - 500.9) ng/ml vs. 213.0 (34.0 - 442.0) ng/ml]および総CSF1R濃度 [820.9 (431.6 - 1415.1) ng/ml vs. 866.0 (516.0 - 1487.6) ng/ml]であった。
　WFA +-CSF1R濃度は、LC患者では肝細胞がん（HCC）罹患の有無で有意差は認めなかったが、総CSF1R濃度は肝硬変患者の中で、HCC合併例は非合併例に比べてわずかに高かった(p = 0.035)。
　WFA+-CSF1R％（総CSF1RにおけるWFA +-CSF1Rの割合）はHCC群と非HCC群で それぞれ26.9％ (11.3- 77.7)及び21.3％ (6.3 - 64.1) (p = 0.0018)となり、HCC群は有意に高値であった。
　またWFA+-CSF1R％は、LC患者はCH患者に比べ有意に高値であった [23.3 (6.3 - 77.7) vs. 15.6 (0.9 - 55.8)] (p ＜ 0.0001) （表１）。
　本発明において対象とするエントリー時にHCCを認めない非HCC肝硬変患者（LC＋HCC－）（56名）について、生存率と累積発がん率を検討した。WFA +-CSF1R 値の中央値は213.0（34.0～442.0）であり、WFA+-CSF1R％値は21.3 (6.3 ～64.1)であった（表２）。 (3-1) WFA + -CSF1R concentration and WFA + -CSF1R% in serum of all patients
Measurement of the WFA + -CSF1R and total CSF1R concentration with serum samples of the entire liver disease patients (214 patients), serum WFA + -CSF1R concentration, the total CSF1R concentration both patients with cirrhosis (LC) (115 patients) is hepatitis [WFA + -CSF1R: 208.9 (34.3-500.9) ng / ml vs. 82.3 (5.0-241.0) ng / ml], [total CSF1R: 845.3 (431.6) -1487.5) ng / ml vs. 536.4 (266.3-1357.2) ng / ml].
Serum WFA + -CSF1R concentration [208.9 (85.4-500.9) ng / ml vs. 213.0 (34.0-442.0) ng / ml] and total CSF1R concentration [820.9 (115 HCC, 59 non-HCC 56) 431.6-1415.1) ng / ml vs. 866.0 (516.0-1487.6) ng / ml].
WFA + -CSF1R levels were not significantly different in LC patients with or without hepatocellular carcinoma (HCC), but total CSF1R levels were slightly higher in patients with cirrhosis than in those without HCC (P = 0.035).
WFA + -CSF1R% (ratio of WFA + -CSF1R in total CSF1R) was 26.9% (11.3-77.7) and 21.3% (6.3-64.1) (p = 0.0018) in the HCC group and non-HCC group, respectively. Significantly higher.
WFA + -CSF1R% was significantly higher in LC patients than in CH patients [23.3 (6.3-77.7) vs. 15.6 (0.9-55.8)] (p <0.0001) (Table 1).
In the present invention, the survival rate and cumulative carcinogenic rate were examined for non-HCC cirrhosis patients (LC + HCC − ) (56 patients) who did not recognize HCC at the time of entry. The median WFA + -CSF1R value was 213.0 (34.0 to 442.0) and the WFA + -CSF1R% value was 21.3 (6.3 to 64.1) (Table 2).
（３－２）全患者におけるWFA+-CSF1R％による発がん率
　まず、WFA+-CSF1Rによる累積発がん率を測定したが有意差がなかった（データ未掲載）。
　一方、CSF1Rは悪性疾患と関連すると言われており、WFA+-CSF1R％と発がんとの関連性を検討した。臨床的因子および癌関連因子を、時間依存性ROC解析で求めたAUCとコックスの回帰分析で求めた危険率（HR）で評価した。本コホートではインターフェロン治療歴およびSVR率は、HCCの進展に影響がなかったため考慮しなかった。累積発生率における診断能は、総観察期間における95％信頼区間のROC曲線下面積（AUC）として示した。連続型変数間の相関はスピアマンの順位相関係数で数量化された。P＜0.05を統計的有意とした。
　肝硬変患者の肝細胞がんの発がん率に対するログランク検定で求められたP値に基づく最小P値法により（上下10%の症例を除外）、発がんを予測するためのWFA+-CSF1R％の最適カットオフ値は35.0％となった（表１）、（図４）。本カットオフ値を用いた生存予測での危険率は1.55（95％ CI 1.03-2.34, p = 0.034）だった。さらに別のサンプルセット（バリデーションセット）を用いてWFA+-CSF1R％の有用性を検討した結果、WFA+-CSF1R％は予測因子として有望であった（HR 4.06, 95％ CI 1.63-10.13, p ＜ 0.001）。
　他のデータと総合してLC患者の累積発がん率に関与する因子を時間依存的ROC分析にて解析したところ、WFA+-CSF1R％はAUC 0.760、HR 1.55（95％CI 1.03-2.34、p = 0.034）であった（表３）。従って、トレーニングセットとバリデーションセットの結果を考慮し、LC患者での累積発がん率との関連を示したWFA+-CSF1R％値35.0％を最適候補とした。さらに、WFA+-CSF1R％と他の連続型変数との相関をスピアマンの順位相関係数検定で分析した結果、相関が認められた（表３）、（図５，６）。 (3-2) Carcinogenicity by WFA + -CSF1R% in all patients First, the cumulative carcinogenicity by WFA + -CSF1R was measured, but there was no significant difference (data not shown).
On the other hand, CSF1R is said to be associated with malignant diseases, and the relationship between WFA + -CSF1R% and carcinogenesis was examined. Clinical factors and cancer-related factors were evaluated by the risk factor (HR) determined by AUC and Cox regression analysis determined by time-dependent ROC analysis. In this cohort, interferon treatment history and SVR rates were not considered because they did not affect the progression of HCC. The diagnostic ability at the cumulative incidence was expressed as the area under the ROC curve (AUC) of the 95% confidence interval during the total observation period. The correlation between continuous variables was quantified by Spearman's rank correlation coefficient. P <0.05 was considered statistically significant.
Optimum WFA + -CSF1R% for predicting carcinogenesis by the minimum P-value method based on the P-value obtained by log-rank test for the incidence of hepatocellular carcinoma in patients with cirrhosis (excluding upper and lower 10% cases) The cut-off value was 35.0% (Table 1) and (Figure 4). The risk factor in predicting survival using this cutoff value was 1.55 (95% CI 1.03-2.34, p = 0.034). As a result of examining the usefulness of WFA + -CSF1R% using another sample set (validation set), WFA + -CSF1R% was promising as a predictor (HR 4.06, 95% CI 1.63-10.13, p <0.001).
In combination with other data, time-dependent ROC analysis of factors involved in cumulative carcinogenicity in LC patients revealed that WFA + -CSF1R% was AUC 0.760, HR 1.55 (95% CI 1.03-2.34, p = 0.034) (Table 3). Therefore, considering the results of the training set and the validation set, the WFA + -CSF1R% value of 35.0%, which showed an association with the cumulative cancer incidence in LC patients, was selected as the best candidate. Furthermore, the correlation between WFA + -CSF1R% and other continuous variables was analyzed by Spearman's rank correlation coefficient test. As a result, a correlation was observed (Table 3) and (FIGS. 5 and 6).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（３－３）肝細胞がんのない肝硬変患者（LC(+)HCC(-)）の肝細胞がん（HCC）発生率について
　そこで、さらに、上記で検討したWFA+-CSF1R％カットオフ値で層別化された患者での発がん率を求めた。LC患者におけるWFA+-CSF1R％と発がん率を明らかにするためHCC患者を二グループに分けた（高値群＞35.0％、低値群＜35.0％。カプラン・マイヤー解析によると、トレーニングセットにおいて、5年の累積発がん率はWFA+-CSF1R％高値群(＞35.0％、6名)で75％、低値群 (＜35.0％、50名)は30％であり、累積発がん率はWFA+-CSF1R％高値群で有意に高かった(P = 0.006)（図７ａ）。同様に バリデーションセットにおいて、5年の累積発がん率はWFA+-CSF1R％高値群(10名)で75％であり、低値群(35名)の42％に比べて累積発がん率は有意に高かった(P = 0.005)（図７ｂ）。
　これらの結果よりWFA+-CSF1R％はHCCに関連があるだけでなく、LC患者の癌化リスク予測マーカーになりうることが示唆された。そして、最小P値法で算出されたWFA+-CSF1R％の値は、LC患者のHCC発がんリスク指数として機能する。 (3-3) Incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis (LC (+) HCC (-)) without hepatocellular carcinoma Therefore, further, the WFA + -CSF1R% cutoff value examined above The carcinogenic rate in patients stratified with To clarify WFA + -CSF1R% and carcinogenesis in LC patients, HCC patients were divided into two groups (high group > 35.0%, low group <35.0%. According to Kaplan-Meier analysis, 5 The cumulative cancer rate is 75% in the WFA + -CSF1R% high group ( > 35.0%, 6 patients) and 30% in the low group (<35.0%, 50 patients), and the cumulative cancer rate is WFA + -CSF1R. (P = 0.006) (Fig. 7a) Similarly, in the validation set, the 5-year cumulative carcinogenic rate was 75% in the WFA + -CSF1R% high group (10 subjects), which was low. The cumulative carcinogenic rate was significantly higher than 42% in the group (35 subjects) (P = 0.005) (FIG. 7b).
These results suggest that WFA + -CSF1R% is not only related to HCC, but may also be a marker for predicting cancer risk in LC patients. The value of WFA + -CSF1R% calculated by the minimum P-value method functions as an HCC carcinogenic risk index for LC patients.
（実施例４）肝細胞がんのない肝硬変患者（LC(+)HCC(-)）の予後（生存率）について
（４－１）全患者におけるWFA+-CSF1R値による生存率
　WFA+-CSF1R値は線維化の進展において上昇するため、WFA+-CSF1RをLC患者の予測因子として評価した。臨床的および癌関連因子を時間依存的ROC曲線のAUC（総観察期間における95％信頼区間のROC曲線下面積）およびコックス回帰分析で計算した危険率で評価した。
　まずトレーニングコホートにおいて、コックス回帰分析で最少のP値を示す値をWFA+-CSF1Rの最適カットオフ値として求めた（310 ng/ml、図８）。生存を予測するための危険率はWFA+-CSF1R値（＞310)において3.63 (95％ CI 1.25-10.54、 p = 0.011)であった。さらにWFA+-CSF1R値の有効性を独立したサンプルセット（バリデーションセットとする）を用いて検討した結果、WFA+-CSF1R値（＞310）は予測因子として有望であった（HR 6.07, 95％ CI 1.62-22.77, p =0.002）。
トレーニングセットとバリデーションセットを総合すると（Table 2）、継続的因子としてAUCは、WFA+-CSF1Rで0.691（HR 1.80, 95％ CI 1.23-2.62, p = 0.002）、アルブミンでは0.719 （HR 0.29, 95％ CI 0.18-0.47, p＜ 0.001）、Fib4では0.706（HR 1.78, 95％ CI 1.25-2.52, p = 0.001）であった。したがってWFA+-CSF1Rは最適な候補であることがわかり、トレーニングセットとバリデーションセットのLC患者予後において最も強い相関を示した。WFA+-CSF1RのLC患者の予後への相関はFib4の相関に類似していたが、本コホートではアルブミンも信頼できる予測因子であった。
　さらに我々は、WFA+-CSF1R値と他の連続変数との相関をスピアマンの順位相関係数検定を用いて調べた。（表４右）に示すようにWFA+-CSF1R値は他の因子に多重相関を見せた。 (Example 4) Prognosis (survival rate) of cirrhosis patients (LC (+) HCC (-)) without hepatocellular carcinoma (4-1) Survival rate by WFA + -CSF1R value in all patients WFA + -CSF1R Since the value increases in the progression of fibrosis, WFA + -CSF1R was evaluated as a predictor of LC patients. Clinical and cancer-related factors were assessed by time-dependent ROC curve AUC (area under the 95% confidence interval ROC curve during the total observation period) and risk calculated by Cox regression analysis.
First, in the training cohort, the value showing the minimum P value in Cox regression analysis was determined as the optimum cutoff value of WFA + -CSF1R (310 ng / ml, FIG. 8). The risk factor for predicting survival was 3.63 (95% CI 1.25-10.54, p = 0.011) for WFA + -CSF1R values ( > 310). Furthermore, as a result of examining the effectiveness of WFA + -CSF1R value using an independent sample set (referred to as a validation set), WFA + -CSF1R value ( > 310) was promising as a predictor (HR 6.07, 95% CI 1.62-22.77, p = 0.002).
Combining the training and validation sets (Table 2), AUC as a continuous factor is 0.691 for WFA + -CSF1R (HR 1.80, 95% CI 1.23-2.62, p = 0.002) and 0.719 for albumin (HR 0.29, 95) % CI 0.18-0.47, p <0.001), and Fib4 was 0.706 (HR 1.78, 95% CI 1.25-2.52, p = 0.001). Therefore, WFA + -CSF1R proved to be the best candidate and showed the strongest correlation between the prognosis for LC patients in the training set and the validation set. The correlation of WFA + -CSF1R to prognosis in LC patients was similar to that of Fib4, but albumin was also a reliable predictor in this cohort.
In addition, we examined the correlation between WFA + -CSF1R values and other continuous variables using Spearman's rank correlation coefficient test. As shown in (Table 4 right), the WFA + -CSF1R value showed multiple correlations with other factors.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（４－２）WFA+-CSF1R値による生存率
　WFA+-CSF1R値は線維化の進展につれて上昇するため、WFA+-CSF1RをLC患者の予後予測因子になりうるかを評価した。LC患者の肝細胞がんの累積生存率に対するログランク検定で求められたP値に基づく最小P値法により（上下10%の症例を除外）、予後を予測するためのWFA+-CSF1Rの最適カットオフ値は310 ng/mlであり（表１）、時間依存的ROC曲線で生存率を検討したところ、WFA+-CSF1R値(＞310)においてHRは3.63 (95％ CI 1.25-10.54、p = 0.011)であった。
　トレーニングセットとバリデーションセットを合わせた結果、WFA+-CSF1R のAUCは0.691（HR 1.80, 95％ CI 1.23-2.62, p = 0.002）であり、WFA+-CSF1RはLC患者における予後予測に良好な結果が得られた（表４）。
　LC患者における累積生存率をカプラン・マイヤー解析にて検討すると、WFA+-CSF1R高値群(＞ 310 ng/ml)の1,3,5年生存率はトレーニングセット（8名）でそれぞれ88％、60％、45％であり、バリデーションセット（10名）で100％、71％、43％であった。一方WFA+-CSF1R低値群(＜ 310 ng/ml）では、トレーニングセット（48名）94％、89％、74％(p= 0.010)、バリデーションセット（35名）で100％、100％、100％ (p ＜ 0.003)（図９ａ，ｂ）であった。WFA+-CSF1R値高値群は低値群に比較し有意に生存率が低かった。 (4-2) WFA + -CSF1R viability WFA + -CSF1R value by value to increase as the progress of fibrosis was evaluated whether can become a WFA + -CSF1R the prognostic factors of LC patients. Optimum WFA + -CSF1R for predicting prognosis by minimum P-value method (excluding upper and lower 10% cases) based on P-value obtained by log rank test for cumulative survival of hepatocellular carcinoma in LC patients The cut-off value was 310 ng / ml (Table 1). When the survival rate was examined with a time-dependent ROC curve, HR was 3.63 (95% CI 1.25-10.54, p for WFA + -CSF1R value ( > 310). = 0.011).
As a result of combining the training set and the validation set, the AUC for WFA + -CSF1R is 0.691 (HR 1.80, 95% CI 1.23-2.62, p = 0.002), and WFA + -CSF1R is a good outcome for predicting prognosis in LC patients Was obtained (Table 4).
When the cumulative survival rate in LC patients was examined by Kaplan-Meier analysis, the 1,3-, 5-year survival rate of the high WFA + -CSF1R group ( > 310 ng / ml) was 88% in the training set (8 subjects), They were 60% and 45%, and 100%, 71% and 43% in the validation set (10 persons). On the other hand, in the WFA + -CSF1R low level group (<310 ng / ml), training set (48 people) 94%, 89%, 74% (p = 0.010), validation set (35 people) 100%, 100%, 100% (p <0.003) (FIGS. 9a and b). The survival rate of the WFA + -CSF1R high group was significantly lower than that of the low group.
　以上の結果から、WFA+-CSF1R値は、LC患者における予後（生存率）因子として優れていることが実証され、最小P値法で算出されたWFA+-CSF1Rの値は、LC患者の予後予測指数として機能する。 These results, WFA + -CSF1R value, it is demonstrated that it is excellent as prognosis (survival) factor in LC patients, the value of the WFA + -CSF1R calculated in the minimum P value method, the prognosis of LC patients Functions as a forecast index.
（実施例５）標準CSF1R糖タンパク質の作製
（５－１）　rCSF1Rの発現系の構築と精製
　肝硬変の血清糖タンパク質マーカーとして開発したWFA+-CSF1Rの微量迅速測定キットの開発において、測定のキャリブレーターとなる糖タンパク質標準品の生産系構築の検討を行った。測定キットは抗体－レクチン サンドイッチ検出系であるため、糖タンパク質標準品には、抗体とレクチンのそれぞれに反応するエピトープが必要となる。標準糖タンパク質作製において、目的の糖鎖を有する糖タンパク質を生産する細胞の選択は非常に重要であると考えられる。WFAに結合する糖鎖を持つ糖タンパク質の発現は、すでに肝線維化マーカーWFA+-M2BPの発現において確立しており、今回もそれに倣いHEK293細胞をタンパク質発現のホスト細胞に用いた。
　CSF1Rタンパク質は972アミノ酸からなる膜タンパク質であり（配列番号１，２）、1-19アミノ酸がシグナル配列、20-517アミノ酸が細胞外領域、518-538アミノ酸が膜貫通領域、539-972アミノ酸が細胞内領域である。細胞外領域には11箇所のN結合型糖鎖付加のコンセンサス配列が存在し、これらのすべてあるいは一部にN結合型糖鎖が結合していることが考えられる。これらの情報を基に、リコンビナントCSF1R(rCSF1R)は、自身のシグナル配列と細胞外領域である1-489アミノ酸をコードする領域をPCRで増幅し、発現ベクターに導入した。
　ヒト単球性白血病細胞株（THP-1）由来cDNAを鋳型に、2本のプライマー、
Fwd: 5’- AGGCCATGGGCCCAGGAGTTCTGCTGCT -3’, （配列番号５）
Rev: 5’- ggaattcGTTGTGGGCCCTGCACTCGTAG -3’（下線はEcoRI部位）（配列番号６）
でPCR反応を行い、増幅した1.5 KbpのDNA断片をpCRII-Bluntベクター（Invitrogen）にサブクローニングし、Genetic Analyzer 3130xl（Applied Biosystems社）で増幅核酸配列を確認した。EcoRIで切断したDNA断片を発現ベクター pcDNA3.1neo(+)DDDDK（Invitrogen社のベクターを改良）のDDDDK tag配列前のEcoRI部位に挿入し、pcDNA3.1-CSF1R-tagを構築した。この結果、このベクターから発現するrCSF1RはC末端にDDDDK tag配列を持つことになる。
　このプラスミドをヒト胎児腎臓由来細胞株HEK293細胞にLipofectamine LTX（Invitrogen）を用いてトランスフェクションし、1 mg/mLのG418（ナカライテスク）を培地に添加して、安定発現株を選別した。構築した安定発現株をDMEM+10％FCS+PS培地でコンフレント状態で48時間培養後の培養上清を3回繰り返し回収し、3100 rpm、10分間の遠心分離の後、上清を回収した。
　回収した培養上清から抗DDDDK抗体カラム（MBL社）を用いてrCSF1Rタンパク質を精製した。0.45μmフィルターろ過した培養上清を、DDDDK抗体カラムに供し、素通りした上清を再度カラムに供した。抗体カラム容量の10倍量の0.1％Tween含有PBS緩衝液で洗浄し、さらに、PBS緩衝液で洗浄を行った。抗体カラム容量の5倍量のDDDDKペプチドを含むPBS緩衝液にて、抗体カラムに結合したrCSF1Rの溶出を行い、さらに限外濾過膜（Amicon 10K）を用いて溶出に用いたDDDDK ペプチドの除去とタンパク質の濃縮を行った。（本発明で得られたrCSF1Rタンパク質を、以下「標準CSF1R（タンパク質）」ともいう。）
　精製したrCSF1Rは、タンパク質濃度を測定した後、-30℃にて保存した。 (Example 5) Preparation of standard CSF1R glycoprotein (5-1) Construction and purification of rCSF1R expression system In the development of a rapid microassay kit for WFA + -CSF1R developed as a serum glycoprotein marker for cirrhosis, The production system of the glycoprotein standard product was examined. Since the measurement kit is an antibody-lectin sandwich detection system, the glycoprotein standard requires an epitope that reacts with each of the antibody and the lectin. In standard glycoprotein production, selection of cells producing a glycoprotein having a target sugar chain is considered to be very important. The expression of a glycoprotein having a sugar chain that binds to WFA has already been established in the expression of liver fibrosis marker WFA + -M2BP. Again, HEK293 cells were used as host cells for protein expression.
CSF1R protein is a membrane protein consisting of 972 amino acids (SEQ ID NOs: 1, 2), 1-19 amino acids are signal sequences, 20-517 amino acids are extracellular regions, 518-538 amino acids are transmembrane regions, and 539-972 amino acids are It is an intracellular region. There are 11 consensus sequences for N-linked sugar chain addition in the extracellular region, and it is considered that N-linked sugar chains are bound to all or part of them. Based on this information, the recombinant CSF1R (rCSF1R) amplified its own signal sequence and a region encoding the extracellular region of 1-489 amino acids by PCR and introduced it into an expression vector.
Using human monocytic leukemia cell line (THP-1) -derived cDNA as a template, two primers,
Fwd: 5'- AGGCCATGGGCCCAGGAGTTCTGCTGCT -3 ', (SEQ ID NO: 5)
Rev: 5'-g gaattc GTTGTGGGCCCTGCACTCGTAG -3 '(underlined EcoRI site) (SEQ ID NO: 6)
The amplified 1.5 Kbp DNA fragment was subcloned into pCRII-Blunt vector (Invitrogen), and the amplified nucleic acid sequence was confirmed with Genetic Analyzer 3130xl (Applied Biosystems). The DNA fragment cleaved with EcoRI was inserted into the EcoRI site before the DDDDK tag sequence of the expression vector pcDNA3.1neo (+) DDDDK (modified from Invitrogen) to construct pcDNA3.1-CSF1R-tag. As a result, rCSF1R expressed from this vector has a DDDDK tag sequence at the C-terminus.
This plasmid was transfected into human fetal kidney-derived cell line HEK293 cells using Lipofectamine LTX (Invitrogen), and 1 mg / mL G418 (Nacalai Tesque) was added to the medium to select stable expression lines. The culture supernatant after culturing the constructed stable expression strain in DMEM + 10% FCS + PS medium in a confluent state for 48 hours was repeatedly collected 3 times, and the supernatant was collected after centrifugation at 3100 rpm for 10 minutes.
The rCSF1R protein was purified from the collected culture supernatant using an anti-DDDDK antibody column (MBL). The culture supernatant filtered through a 0.45 μm filter was applied to a DDDDK antibody column, and the passed supernatant was applied to the column again. Washing was performed with PBS buffer solution containing 0.1% Tween, 10 times the volume of the antibody column, and further with PBS buffer solution. Elution of rCSF1R bound to the antibody column with PBS buffer containing 5 times the volume of the antibody column DDDDK peptide, and removal of the DDDDK peptide used for elution using an ultrafiltration membrane (Amicon 10K) Protein concentration was performed. (The rCSF1R protein obtained in the present invention is hereinafter also referred to as “standard CSF1R (protein)”.)
The purified rCSF1R was stored at −30 ° C. after measuring the protein concentration.
（５－２）　標準CSF1R（rCSF1R）の糖鎖プロファイリング
　抗体オーバーレイ・レクチンマイクロアレイ法
　レクチンマイクロアレイのプラットフォームは基本的に上記の通りとし、検出に際しては上記被験者を直接蛍光等で標識するのではなく、抗体を介して間接的に蛍光基等を被験者に導入することで、一斉に多検体に対する分析を簡便、高速化することができる応用法である（「Kuno A, Kato Y, Matsuda A, Kaneko MK, Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi J. Mol Cell Proteomics. 8, 99-108(2009)」、「平林淳、久野敦、内山昇「レクチンマイクロアレイを用いた糖鎖プロファイリング応用技術の開発」、実験医学増刊「分子レベルから迫る癌診断研究～臨床応用への挑戦～」、羊土社、Vol25(17)164-171(2007)」、久野敦、平林淳「レクチンマイクロアレイによる糖鎖プロファイリングシステムの糖鎖バイオマーカー探索への活用」、遺伝子医学MOOK11号「臨床糖鎖バイオマーカーの開発と糖鎖機能の解明」、pp.34-39、メディカルドゥ（2008）参照）。
　例えば、糖タンパク質が被検物質であれば糖鎖部分はレクチンマイクロアレイ上のレクチンによって認識されるため、コアタンパク質部分に対する抗体をその上から被せる（オーバーレイ）ことによって、被検糖タンパク質を標識したり、あるいは高度に精製したりすることなく、特定的に感度高く検出することができる。 (5-2) Sugar chain profiling of standard CSF1R (rCSF1R) Antibody overlay / lectin microarray method The lectin microarray platform is basically as described above, and the subject is not directly labeled with fluorescence or the like for detection. This is an application method that allows simultaneous analysis of multiple specimens to be simplified and accelerated by introducing fluorescent groups etc. into the subject indirectly via (Kuno A, Kato Y, Matsuda A, Kaneko MK, Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi J. Mol Cell Proteomics. "Development", Experimental medicine special edition "Cancer diagnostic research approaching from molecular level-Challenge to clinical application", Yodosha, Vol25 (17) 164-171 (2007) ", Satoshi Kuno, Satoshi Hirabayashi" Glycan by lectin microarray Professional Utilization of filing system for glycan biomarker search ”, Gene Medicine MOOK11“ Development of clinical glycan biomarker and elucidation of glycan function ”, pp.34-39, Medical Do (2008)).
For example, if the glycoprotein is a test substance, the sugar chain part is recognized by the lectin on the lectin microarray, so that the test glycoprotein can be labeled by overlaying the antibody against the core protein part (overlay). Alternatively, it can be detected specifically with high sensitivity without being highly purified.
　具体的には下記の通り行った。
　リコンビナントCSF1R糖タンパク質の糖鎖プロファイルを解析するために、抗体オーバーレイ・レクチンマイクロアレイ法による解析を行った。
レクチンマイクアロアレイは、45種の異なるレクチンが3スポットずつ固定化されているものを使用した(LecChipTM、株式会社グライコテクニカ)。上記アレイに、緩衝液で希釈したLDN陽性とLDN陰性のリコンビナントCSF1Rをそれぞれ200ng/wellアプライし、緩やかに振盪しながら20℃で12時間レクチンへの結合反応を行った。反応後、ヒトIgGを2μg/well添加し30分間ブロッキングを行った。次にアレイ上のブロッキング剤を含む試料溶液を除去し、専用の緩衝液で3回洗浄した後、20ug/mLヒトIgGを含む緩衝液で100倍に希釈したビオチン化抗CSF1Rポリクローナル抗体（R&D Systems）を添加し、緩やかに振盪しながら20℃で1時間抗体の結合反応を行った。反応後、抗体溶液を除去し専用緩衝液で3回洗浄した後、緩衝液で5000倍に希釈したCy3結合ストレプトアビジン（GEヘルスケア）を添加し、緩やかに振盪しながら20℃で1時間二次反応を行った。 反応後、二次反応液を除去し専用緩衝液で3回洗浄した後、株式会社グライコテクニカ製レクチンマイクロアレイ用スキャナー（GlycoStationTM Reader 1200）を用いてシグナル強度を測定した。バックグラウンド値を引いた真値算出後、各レクチン3スポット間の平均値を算出し、全レクチンで最大のシグナル強度を基準値と定めて、相対値を求め、数値化を行った（図１０）。 Specifically, it was performed as follows.
In order to analyze the sugar chain profile of the recombinant CSF1R glycoprotein, the antibody overlay / lectin microarray analysis was performed.
The lectin microphone alloarray used was one in which 45 different lectins were immobilized at 3 spots each (LecChip ™ , Glyco Technica Co., Ltd.). 200 ng / well of LDN-positive and LDN-negative recombinant CSF1R diluted with a buffer solution was applied to the above array, and a lectin binding reaction was performed at 20 ° C. for 12 hours while gently shaking. After the reaction, human IgG was added at 2 μg / well and blocked for 30 minutes. Next, the sample solution containing the blocking agent on the array was removed, washed with a dedicated buffer three times, and then biotinylated anti-CSF1R polyclonal antibody (R & D Systems) diluted 100 times with a buffer containing 20 ug / mL human IgG. ) And the antibody was allowed to bind for 1 hour at 20 ° C. with gentle shaking. After the reaction, remove the antibody solution and wash 3 times with the dedicated buffer. Then add Cy3-conjugated streptavidin (GE Healthcare) diluted 5000 times with the buffer solution, and gently shake at 20 ° C for 1 hour. The next reaction was performed. After the reaction, the secondary reaction solution was removed and washed with a dedicated buffer three times, and then the signal intensity was measured using a scanner for lectin microarray (GlycoStation ™ Reader 1200) manufactured by Glyco Technica Co., Ltd. After calculating the true value by subtracting the background value, the average value between the three spots of each lectin was calculated, the maximum signal intensity of all lectins was determined as the reference value, the relative value was obtained, and the quantification was performed (FIG. 10). ).
（５－３）rCSF1Rの糖鎖構造解析
　上記の精製したrCSF1Rを用いて、IGOT法にてN結合型糖鎖付加部位を決定し、さらにGlycoRidge法によりN結合型糖鎖付加部位ごとに糖鎖構造解析を行った。
　ここで、GlycoRidge法は、本発明者らが開発したリコンビナントタンパク質のペプチド配列ならびに糖鎖構造の解析技術（Noro E, et al, J Proteome Res. 2015 Sep 4;14(9):3823-34.）であり、概ね以下の手順で行う。
　リコンビナント糖タンパク質をDTTおよびヨードアセトアミドにより還元アルキル化し、トリプシン消化後、回収した糖タンパク質を酸性条件下（pH2以下）で高温加熱（例えば、80℃、2時間加熱）しシアル酸を切除する。この糖ペプチドをLC/MS法で分析し、個々の糖ペプチドシグナルの精密質量をリスト化する。糖鎖付加部位を含むペプチドの計算質量と、観測した糖ペプチドの質量差から、糖鎖部分の単糖組成を推定し、推定された糖鎖モチーフを含むと推定される付加部位を同定する。糖ペプチドから遊離した糖鎖をMALDI-TOF MSで分析し、推定糖鎖モチーフを含有すると思われる組成の糖鎖から、推定糖鎖モチーフに対応する断片イオンが検出されれば推定糖鎖モチーフ及びその付加位置（ペプチド配列）が正しかったことが確認できる。 (5-3) Sugar chain structure analysis of rCSF1R Using the purified rCSF1R described above, N-linked glycosylation sites are determined by the IGOT method, and each N-linked glycosylation site is determined by the GlycoRidge method. Structural analysis was performed.
Here, the GlycoRidge method is a technique for analyzing the peptide sequence and sugar chain structure of the recombinant protein developed by the present inventors (Noro E, et al, J Proteome Res. 2015 Sep 4; 14 (9): 3823-34. ) And generally follow the procedure below.
Recombinant glycoprotein is reductively alkylated with DTT and iodoacetamide, and after digestion with trypsin, the recovered glycoprotein is heated at high temperature (for example, 80 ° C., 2 hours) under acidic conditions (pH 2 or lower) to excise sialic acid. The glycopeptide is analyzed by LC / MS method and the exact mass of each glycopeptide signal is listed. The monosaccharide composition of the sugar chain portion is estimated from the calculated mass of the peptide containing the glycosylation site and the mass difference between the observed glycopeptides, and the addition site presumed to contain the estimated sugar chain motif is identified. The glycan released from the glycopeptide is analyzed by MALDI-TOF MS, and if a fragment ion corresponding to the putative glycan motif is detected from the glycan having a composition that seems to contain the putative glycan motif, It can be confirmed that the addition position (peptide sequence) was correct.
　具体的には以下の手順で行った。
　組換え体CSF1Rを還元アルキル化（タンパク質と等重量のDTT、およびヨードアセトアミド （タンパク質×2.5倍重量）で反応させ、その後透析するなどの常法に従う）の後、トリプシン消化し、アミド80カラム（東ソー株式会社: TOSOH）で糖ペプチドを回収した。これを酸性条件下（pH～2)、80℃、2時間加熱し、シアル酸を切除した。この糖ペプチドをLC/MS法で分析し、個々の糖ペプチドシグナルの精密質量をリスト化した（誤差は2ppm以下：サーモサイエンティフィック社LTQ-Orbitrap Velos）。
　糖鎖付加部位を含むペプチドの計算質量と、観測した糖ペプチドの質量差から、糖鎖部分の単糖組成(Hex)*i+(HexNAc)*j+(dHex)*k; Hex=Man/Gal, HexNAc=GlcNAc/GalNAc, dHex=Fucoseを推定した。推定された糖鎖組成（たとえばHex4+HexNAc5+Fuc1)からGalNAc-GlcNAc（=LacDiNAc）モチーフを含むと推定される付加部位を同定した。糖ペプチドから遊離した糖鎖をMALDI-TOF MSで分析し、LacDiNAcを含有すると思われる組成の糖鎖（以下、LDN糖鎖ともいう）から、HexNAc-HexNAc断片イオンが検出され、その存在を確認した。 Specifically, the procedure was as follows.
Recombinant CSF1R is subjected to reductive alkylation (reacted with DTT of equal weight of protein and iodoacetamide (protein × 2.5 times weight) and then dialyzed) followed by trypsin digestion and amide 80 column ( Tosoh Corporation: TOSOH) recovered the glycopeptide. This was heated under acidic conditions (pH˜2) at 80 ° C. for 2 hours to excise sialic acid. This glycopeptide was analyzed by LC / MS method, and the exact mass of each glycopeptide signal was listed (error is 2 ppm or less: Thermo Scientific LTQ-Orbitrap Velos).
From the calculated mass of the peptide containing the glycosylation site and the mass difference of the observed glycopeptide, the monosaccharide composition of the glycan moiety (Hex) * i + (HexNAc) * j + (dHex) * k; Hex = Man / Gal, HexNAc = GlcNAc / GalNAc, dHex = Fucose were estimated. From the estimated sugar chain composition (for example, Hex4 + HexNAc5 + Fuc1), an additional site presumed to contain a GalNAc-GlcNAc (= LacDiNAc) motif was identified. The glycan released from the glycopeptide is analyzed by MALDI-TOF MS, and the HexNAc-HexNAc fragment ion is detected from the glycan of the composition that seems to contain LacDiNAc (hereinafter also referred to as LDN glycan) and its presence is confirmed. did.
　その結果、市販CSF1Rタンパク質（図１１）および標準CSF1R糖タンパク質（図１２）における糖ペプチドを同定し、糖鎖の付加位置を明らかにした（図１３）。標準CSF1R糖タンパク質では73Asnおよび153Asnの糖鎖にWFA陽性となる（エピトープとなる）LacdiNAc構造（LDN糖鎖）と結合することを確認した。 As a result, glycopeptides in the commercially available CSF1R protein (FIG. 11) and the standard CSF1R glycoprotein (FIG. 12) were identified, and the sugar chain addition position was clarified (FIG. 13). It was confirmed that the standard CSF1R glycoprotein binds to the 73Asn and 153Asn sugar chains with a LacdiNAc structure (LDN sugar chain) that is WFA positive (becomes an epitope).
（実施例６）LDN欠損株を用いた組換えCSF1R上のLDN糖鎖位置の確認
（６－１）LDN欠損株の構築
　（実施例５）の（５－１）で得られたrCSF1Rを産生する形質転換HEK293細胞に対してLDN糖鎖特異的な転移活性を有する糖転移酵素であるB4GALNT3及びB4GALNT4をコードする遺伝子に、CRISPR/Cas9システム（Jennifer A. Doudna, et al., Science 28 Nov 2014:Vol. 346, Issue 6213,DOI: 10.1126/science.1258096）で不活性型変異を導入することによりLDN欠損株を作製し、LDN糖鎖（WFAの結合する糖鎖）を欠損した標準CSF1R糖タンパク質を産生させた。
　具体的には、LDNが発現しているHEK293細胞において、Invitrogen社のGeneArt CRISPR Nuclease Vector Kitを用いて、B4GALNT3、B4GALNT4遺伝子に連続して変異を導入した。B4GALNT3への変異の導入のために、exon2に設計した（配列番号７）に示される標的配列をGeneArt CRISPR Nuclease CD4 Vectorにクローニングしたプラスミドを構築し、Lipofectamine LTX (Invitrogen) を用いてHEK293細胞にトランスフェクションした。24～48時間後、CD4 Enrichment Kit (Invitrogen) を用いて、プラスミドが導入された細胞を選別し、限界希釈法により複数のシングルクローンを単離した。単離したシングルクローン株のゲノムDNAから（配列番号８）及び（配列番号９）に示される塩基配列からなるプライマーセットで標的部位周辺の配列をPCRで増幅し、塩基解読して、3000bpからなるB4GALNT3遺伝子のコーディング領域の209番目にアデニン挿入によるフレームシフト変異が導入されていることを確認した。
　続いて、B4GALNT4への変異の導入のために、exon2に設計した（配列番号１０）に示される標的配列をGeneArt CRISPR Nuclease CD4 Vectorにクローニングしたプラスミドを構築し、Lipofectamine LTX を用いてB4GALNT3変異細胞にトランスフェクションした。同様にCD4陽性のシングルクローンを単離の後、（配列番号１１）及び（配列番号１２）に示される塩基配列からなるプライマーセットで標的部位周辺の配列をPCRで増幅し、塩基解読して、3120bpからなるB4GALNT4遺伝子のコーディング領域の184番目のシトシン欠失によるフレームシフト変異が導入されていることを確認した。 (Example 6) Confirmation of LDN sugar chain position on recombinant CSF1R using LDN-deficient strain (6-1) Construction of LDN-deficient strain Production of rCSF1R obtained in (5-1) of (Example 5) CRISPR / Cas9 system (Jennifer A. Doudna, et al., Science 28 Nov 2014), which encodes B4GALNT3 and B4GALNT4, which are glycosyltransferases having LDN sugar chain-specific transfer activity for transformed HEK293 cells : Vol. 346, Issue 6213, DOI: 10.1126 / science.1258096), an LDN-deficient strain was prepared by introducing an inactive mutation, and the standard CSF1R sugar lacking the LDN sugar chain (the sugar chain to which WFA binds) Protein was produced.
Specifically, in HEK293 cells expressing LDN, mutations were successively introduced into the B4GALNT3 and B4GALNT4 genes using Invitrogen's GeneArt CRISPR Nuclease Vector Kit. In order to introduce mutations into B4GALNT3, a plasmid was constructed by cloning the target sequence shown in exon2 (SEQ ID NO: 7) into GeneArt CRISPR Nuclease CD4 Vector, and transferred to HEK293 cells using Lipofectamine LTX (Invitrogen). Erected. After 24-48 hours, the plasmid-introduced cells were selected using CD4 Enrichment Kit (Invitrogen), and a plurality of single clones were isolated by limiting dilution. The sequence around the target site is amplified by PCR with the primer set consisting of the base sequences shown in (SEQ ID NO: 8) and (SEQ ID NO: 9) from the genomic DNA of the isolated single clone strain, and the base is decoded to comprise 3000 bp It was confirmed that a frameshift mutation due to adenine insertion was introduced at position 209 of the coding region of the B4GALNT3 gene.
Subsequently, in order to introduce mutations into B4GALNT4, a plasmid was constructed by cloning the target sequence shown in exon2 (SEQ ID NO: 10) into GeneArt CRISPR Nuclease CD4 Vector, and Lipofectamine LTX was used to construct B4GALNT3 mutant cells. Transfected. Similarly, after isolating a CD4 positive single clone, the sequence around the target site was amplified by PCR with a primer set consisting of the base sequences shown in (SEQ ID NO: 11) and (SEQ ID NO: 12), and the base was decoded. It was confirmed that a frameshift mutation due to deletion of cytosine at position 184 in the coding region of the B4GALNT4 gene consisting of 3120 bp was introduced.
（６－２）LDN欠損株産生rCSF1R上の糖鎖構造との比較による、LDN結合位置の確認
　（実施例３）の（３－３）と同様の手法で、LDN欠損株が産生する変異rCSF1R上の糖鎖構造とその糖鎖位置を決定し（図１３）、rCSF1Rの73位及び153位の位置に結合した糖鎖中に、標準CSF1RではLDN糖鎖が含まれるが、市販CSF1R(NS0)では失われていることが確認できた。 (6-2) Confirmation of LDN binding position by comparison with the sugar chain structure on LCSF-deficient strain-produced rCSF1R In the same manner as (3-3) in (Example 3), mutant rCSF1R produced by LDN-deficient strain The above sugar chain structure and the position of the sugar chain are determined (FIG. 13). Among the sugar chains bound at positions 73 and 153 of rCSF1R, the standard CSF1R contains an LDN sugar chain, but the commercially available CSF1R (NS0 ) Confirmed that it was lost.
（実施例７）抗CSF1R抗体の作製
（７－１）CSF1Rタンパク質のマウスへの免疫
　市販のリコンビナントCSF1Rタンパク質（R&D Systems Inc. 329-MR-100）をマウス（Balb/cマウス、8週齢のメス）に免疫した。生理食塩水に溶解したCSF1Rタンパク質を完全フロイント アジュバントと混合し、これを初日（Day 0）に50 μg分、Day 14に25 μg分、Day 29に25 μg分、Day 42に25 μg分、Day 66に10 μg分を腹腔注射して免疫した。マウスの眼窩採血を定期的に行い、血清中の抗原に対する抗体価の上昇をモニタリングしながら免疫を行った。
　抗体価が十分に上昇したことを確認した免疫マウスから抗体産生細胞を採取した。採取は、最終免疫の日から2～5日後が好ましいため、3日後に採取した。抗体産生細胞としては、脾細胞、リンパ節細胞、末梢血細胞等が挙げられるが、脾細胞又は局所リンパ節細胞が好ましい。マウスからの抗体産生細胞の採取方法は、当該分野で公知の技術に従って行えばよい。そこで、脾細胞を採取し、後述の融合作業を行った。 (Example 7) Preparation of anti-CSF1R antibody (7-1) Immunization of mice with CSF1R protein Commercially available recombinant CSF1R protein (R & D Systems Inc. 329-MR-100) was used in mice (Balb / c mice, 8 weeks old). The female was immunized. CSF1R protein dissolved in physiological saline is mixed with complete Freund's adjuvant, and this is mixed with 50 μg for the first day (Day 0), 25 μg for Day 14, 25 μg for Day 29, 25 μg for Day 42, Day 66 was immunized by intraperitoneal injection of 10 μg. Orbital blood sampling was performed on mice periodically, and immunization was performed while monitoring the increase in antibody titer against the antigen in the serum.
Antibody-producing cells were collected from immunized mice that were confirmed to have sufficiently increased antibody titers. Since the collection was preferably 2 to 5 days after the last immunization, it was collected 3 days later. Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, and spleen cells or local lymph node cells are preferred. A method for collecting antibody-producing cells from mice may be performed according to a technique known in the art. Therefore, spleen cells were collected and a fusion operation described later was performed.
（７－２）ハイブリドーマの作製
　続いて、抗体産生細胞と骨髄腫（ミエローマ）細胞との細胞融合を行うことで、抗CSF1Rモノクローナル抗体を生産するハイブリドーマを作製することができる。
　免疫マウス由来脾細胞とマウス骨髄腫細胞（P3U1細胞）を使用して、常法（後述）に従い、それぞれの細胞をRPMI培地で洗浄した後に混合し、細胞融合促進剤（PEG1500）による細胞融合作業を行った。脾細胞とマウス骨髄腫細胞（P3U1）の混合比率は8：1にて行った。細胞融合後、選択培地としてHAT培地（RPMI1640培地に100単位/mLペニシリン、100 μg/mLストレプトマイシン及び10％ 牛胎児血清(FBS)、10-4Mヒポキサンチン、1.5×10-5Mチミジン及び4×10-7Mアミノプテリンを加えた培地）にて培養を行い、融合細胞のみが生存するように選択的な培養を行った。選択培地で培養開始後約10日以降に生育してくる細胞をハイブリドーマとして選択するため、次に限界希釈法によって、モノクローンの細胞を得た。具体的には、96穴培養プレートに細胞溶液（濃度）を濃いものから薄いものへと希釈系列を作製するようにして播種し、数の限定された細胞由来のハイブリドーマ細胞群を選択するとともに、後述のスクリーニングによってCSF1Rに対する抗体を産生するクローン（を含む96ウェルプレートの陽性ウェル）を選択した。 (7-2) Production of Hybridoma Subsequently, a hybridoma producing an anti-CSF1R monoclonal antibody can be produced by cell fusion of antibody-producing cells and myeloma (myeloma) cells.
Using immunized mouse-derived spleen cells and mouse myeloma cells (P3U1 cells), in accordance with the usual method (described later), each cell is washed with RPMI medium and then mixed, and cell fusion with a cell fusion promoter (PEG1500) Went. The mixing ratio of splenocytes and mouse myeloma cells (P3U1) was 8: 1. After cell fusion, HAT medium (RPMI1640 medium with 100 units / mL penicillin, 100 μg / mL streptomycin and 10% fetal bovine serum (FBS), 10-4 M hypoxanthine, 1.5 × 10 −5 M thymidine and 4 × after cell fusion. The cells were cultured in a medium supplemented with 10 −7 M aminopterin) and selectively cultured so that only the fused cells survived. Monoclonal cells were then obtained by a limiting dilution method in order to select cells that grow after about 10 days from the start of culture in a selective medium as hybridomas. Specifically, the cell solution (concentration) was seeded in a 96-well culture plate so as to prepare a dilution series from a thicker one to a thinner one, and a limited number of cell-derived hybridoma cell groups were selected, A clone that produced an antibody against CSF1R (including positive wells of a 96-well plate) was selected by screening described below.
　スクリーニング方法は以下の通りである。
　増殖してきたハイブリドーマの培養上清中に、目的とする抗CSF1Rモノクローナル抗体が含まれる否かを酵素免疫測定法（ELISA法）によりスクリーニングした。ハイブリドーマを培養したウェル中に含まれる培養上清の一部を採取し、免疫原として使用したCSF1Rリコンビナントタンパク質に対する結合活性を指標とした。CSF1Rリコンビナントタンパク質を96ウェルプレートに固相化（1 μg/mLで100 μL/well）し、ブロッキング後、培養上清を100 μL入れて37度にて1時間反応させた。ELISAによるスクリーニングと限界希釈法（具体的には、96穴培養プレートに1ウェルあたり0.3個程度の細胞が含まれる濃度にて播種した）にて、陽性のクローンを選択していった。一次スクリーニング時には205ウェルの陽性ウェルがあり、これを展開してさらに絞り込み、二次スクリーニング時は100ウェル、そして最終的に抗CSF1Rモノクローナル抗体産生細胞であるハイブリドーマを33クローン選抜した。
　最終的に上記スクリーニング法によって選抜された33クローンの抗CSF1Rモノクローナル抗体産生ハイブリドーマを、それぞれCSR-1～CSR-33と命名した（表５）。 The screening method is as follows.
It was screened by enzyme immunoassay (ELISA method) whether or not the target anti-CSF1R monoclonal antibody was contained in the culture supernatant of the hybridoma that had proliferated. A part of the culture supernatant contained in the well in which the hybridoma was cultured was collected, and the binding activity to the CSF1R recombinant protein used as an immunogen was used as an index. CSF1R recombinant protein was immobilized on a 96-well plate (100 μL / well at 1 μg / mL), blocked, and 100 μL of culture supernatant was added and reacted at 37 ° C. for 1 hour. Positive clones were selected by ELISA screening and limiting dilution (specifically, seeded at a concentration containing about 0.3 cells per well in a 96-well culture plate). There were 205 positive wells at the time of the primary screening, which were expanded and further narrowed down, 100 wells at the time of the secondary screening, and finally 33 clones of hybridomas that were anti-CSF1R monoclonal antibody producing cells were selected.
The 33 clones of anti-CSF1R monoclonal antibody-producing hybridomas finally selected by the screening method were named CSR-1 to CSR-33, respectively (Table 5).
　抗体の精製を行うために、得られたハイブリドーマ細胞の培養上清を100 mL～1 L程度調整した。これをプロテインGが固相化されたカラムを使用したアフィニティークロマトグラフィー法によって精製を行った。 In order to purify the antibody, the culture supernatant of the obtained hybridoma cells was adjusted to about 100 to 1 L. This was purified by affinity chromatography using a column on which protein G was immobilized.
（７－３）ハイブリドーマの寄託
　これらのクローンうち、典型的なタイプの抗CSF1Rモノクローナル抗体CSR-3、CSR-4、CSR-18、CSR-21及びCSR-30を産生するハイブリドーマは、CSR-3（受託番号：NITE BP-02117）、あるいはCSR-4（受託番号：NITE BP -02118）CSR-18（受託番号：NITE BP -02119）CSR-21（受託番号：NITE BP -02120）CSR-30（受託番号：NITE BP -02121）として、2015年9月10日付で独立行政法人　製品評価技術基盤機構（NITE）特許微生物寄託センター(NPMD)に寄託された後、2016年9月7日付で国際寄託に移管された。
　これらのハイブリドーマ細胞株は、RPMI1640に10％FBSを添加した培地を用いて37℃で好適に培養することができる。
　また、抗CSF1Rモノクローナル抗体は、慣用的技術によって回収可能であり、抗体の精製が必要な場合には、イオン交換クロマトグラフィー法、プロテインA又はプロテインG等によるアフィニティークロマトグラフィー法、ゲルクロマトグラフィー法、硫酸アンモニウム塩析法等公知の方法を用いて精製することができる。 (7-3) Deposit of hybridoma Among these clones, hybridomas producing typical types of anti-CSF1R monoclonal antibodies CSR-3, CSR-4, CSR-18, CSR-21 and CSR-30 are CSR-3. (Accession number: NITE BP-02117) or CSR-4 (Accession number: NITE BP-02118) CSR-18 (Accession number: NITE BP-02119) CSR-21 (Accession number: NITE BP-02120) CSR-30 (Deposit number: NITE BP-02121), deposited on September 10, 2015 at the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (NPMD), and then on September 7, 2016 It was transferred to the deposit.
These hybridoma cell lines can be suitably cultured at 37 ° C. using a medium in which 10% FBS is added to RPMI1640.
Further, the anti-CSF1R monoclonal antibody can be recovered by a conventional technique. When purification of the antibody is required, ion exchange chromatography, affinity chromatography using protein A or protein G, gel chromatography, It can refine | purify using well-known methods, such as an ammonium sulfate salting-out method.
（実施例８）各抗CSF1R抗体の性能評価
（８－１）ウェスタンブロット法による生化学的解析
　抗CSF1R抗体を使用して、該分子のウェスタンブロット法による検出を行った。ウェスタンブロット法は一般的な方法に従った。まず、（図１４左）に示すように、CSF1R(M-CSFR)ほかサンプルをSDS-PAGE還元条件下で10％ポリアクリルアミドゲルを用いて電気泳動し、PVDF膜に転写した。5％スキムミルク含有PBSでブロッキング後、一次抗体（抗CSF1R抗体の各クローン）と室温にて1時間反応させた。PVDF膜の洗浄後、二次抗体（0.5μg/mLのHRPラベル化抗マウスIgG抗体）と室温で1時間反応させた。これらのPVDF膜を洗浄後ウェスタンブロッティング検出試薬（Perkin Elmer社）により化学発光にて検出した。 (Example 8) Performance evaluation of each anti-CSF1R antibody (8-1) Biochemical analysis by Western blotting The anti-CSF1R antibody was used to detect the molecule by Western blotting. The Western blot method followed a general method. First, as shown in FIG. 14 (left), CSF1R (M-CSFR) and other samples were electrophoresed using 10% polyacrylamide gel under SDS-PAGE reducing conditions and transferred to a PVDF membrane. After blocking with PBS containing 5% skim milk, the mixture was reacted with a primary antibody (each clone of anti-CSF1R antibody) at room temperature for 1 hour. After washing the PVDF membrane, it was reacted with a secondary antibody (0.5 μg / mL HRP-labeled anti-mouse IgG antibody) at room temperature for 1 hour. These PVDF membranes were washed and detected by chemiluminescence using a Western blotting detection reagent (Perkin Elmer).
（結果）
　結果を図１４右表に示す。サンプルとしてM-CSFR (CSF1R)および細胞株THP-1の培養上清、そして、陰性コントロールとしてのヒトIgG、His-Tag融合タンパク質、ウシ血清に対する反応性を確認した。その結果、18クローンに反応性が認められ、これらがCSF1Rに対するモノクローナル抗体であることが示された。また、各クローンについて（表５）に結果をまとめたものを示しておく。 (result)
The results are shown in the right table of FIG. The reactivity with respect to the culture supernatant of M-CSFR (CSF1R) and the cell line THP-1 as samples, and human IgG, His-Tag fusion protein, and bovine serum as negative controls was confirmed. As a result, reactivity was observed in 18 clones, indicating that these were monoclonal antibodies against CSF1R. In addition, a summary of the results is shown in Table 5 for each clone.
（８－２）各抗体クローンの認識する抗原エピトープ領域の解析
性能を評価するのにあたり、標準糖タンパク質としてさらに種類を拡充した。これらの糖タンパク質は、（図１５）に示す通り、ドメインを削り込んで短くしたタンパク質として作製した。これらの各標準糖タンパク質に対する反応性をウェスタンブロット法によって検討した。ウェスタンブロット法は一般的な方法に従った。まず、各標準糖タンパク質サンプルをSDS-PAGE還元条件下で10％ポリアクリルアミドゲルを用いて電気泳動し、PVDF膜に転写した。5％スキムミルク含有PBSでブロッキング後、一次抗体（抗CSF1R抗体の各モノクローナル抗体クローン）と室温にて1時間反応させた。PVDF膜の洗浄後、二次抗体（0.5μg/mLのHRPラベル化抗マウスIgG抗体）と室温で1時間反応させた。これらのPVDF膜を洗浄後ウェスタンブロッティング検出試薬（Perkin Elmer社）により化学発光にて検出した（図１６）。この解析の結果、（図１７）に示す通り、各クローンの産生する抗体がどのタンパク質領域に反応するのかが明らかになった。 (8-2) In evaluating the analysis performance of the antigen epitope region recognized by each antibody clone, the types of standard glycoproteins were further expanded. As shown in FIG. 15, these glycoproteins were prepared as proteins with domains shortened. The reactivity to each of these standard glycoproteins was examined by Western blotting. The Western blot method followed a general method. First, each standard glycoprotein sample was electrophoresed on a 10% polyacrylamide gel under SDS-PAGE reducing conditions and transferred to a PVDF membrane. After blocking with PBS containing 5% skim milk, the mixture was reacted with the primary antibody (each monoclonal antibody clone of anti-CSF1R antibody) at room temperature for 1 hour. After washing the PVDF membrane, it was reacted with a secondary antibody (0.5 μg / mL HRP-labeled anti-mouse IgG antibody) at room temperature for 1 hour. These PVDF membranes were washed and then detected by chemiluminescence using a Western blotting detection reagent (Perkin Elmer) (FIG. 16). As a result of this analysis, as shown in (FIG. 17), it was revealed to which protein region the antibody produced by each clone reacts.
（実施例９）本発明で得られた抗CSF1R抗体を用いた抗体-抗体 ELISA測定系
（９－１）抗体-抗体 ELISA測定系による総CSF1R分子の検出
　抗CSF1Rモノクローナル抗体を使用して、該分子（総CSF1R分子）の抗体-抗体 ELISA測定系による検出を行った。樹立した抗CSF1Rモノクローナル抗体をそれぞれELISAプレートに固相化し、検出側には市販の抗CSF1Rポリクローナル抗体を用いてELISA測定系への利用の可否についての検討を行った。抗体の組み合わせは一般的にELISAプレート固相化側でも検出側（液相側）でも、どちらに使用しても良く、感度が高くなる抗体の組み合わせにて検出系の構築を行う。一般的には感度が高く、バックグラウンドとなるノイズが少なくなる組み合わせにて検出系の構築を行う。
　まず、各抗体をPBSで4μg/mLとなるように希釈し、ELISA用マイクロプレートに100uL/ウェルずつ添加した。4℃で一晩各抗体をプレートに吸着させた後、溶液を廃棄して、ウェルをPBS-T (PBS, 0.05％ Tween-20)洗浄した。次に、ブロッキング液(PBS with 3％ BSA)を300μL/ウェルで加えて、ブロッキングをした。前記ブロッキング液を廃棄し、洗浄した後、サンプル（CSF1Rタンパク質：R&D Recombinant Human M-CSFR Fc Chimera Cat#329-MR-100）の溶液100μLを各ウェルに添加した。37℃で2時間反応させた後、ウェル中の溶液を廃棄し、PBS-Tにて洗浄した後、ビオチン標識抗CSF1R抗体（R&D biotinylated anti-CSF1R pAb Cat#BAF329）を2μg/mLに調製して、室温で1時間反応させた．その後、溶液を廃棄して洗浄後、西洋ワサビペルオキシダーゼ（HRP）標識ストレプトアビジン（Jackson社）溶液を1ウェルに100μL加えて1時間室温にて反応させた。反応液を廃棄、洗浄した後、1StepUltra TMB基質液（Pierce社）による発色を450nmの吸光度で測定した。 (Example 9) Antibody-antibody ELISA measurement system using anti-CSF1R antibody obtained in the present invention (9-1) Antibody-antibody Detection of total CSF1R molecule by ELISA measurement system Using anti-CSF1R monoclonal antibody, Molecules (total CSF1R molecules) were detected by an antibody-antibody ELISA measurement system. Each established anti-CSF1R monoclonal antibody was immobilized on an ELISA plate, and a commercially available anti-CSF1R polyclonal antibody was used on the detection side to examine whether it could be used in an ELISA measurement system. In general, the combination of antibodies may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and a detection system is constructed with a combination of antibodies with high sensitivity. In general, a detection system is constructed with a combination of high sensitivity and low background noise.
First, each antibody was diluted with PBS to 4 μg / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 μL / well for blocking. After the blocking solution was discarded and washed, 100 μL of a sample solution (CSF1R protein: R & D Recombinant Human M-CSFR Fc Chimera Cat # 329-MR-100) was added to each well. After reacting at 37 ° C for 2 hours, discard the solution in the well and wash with PBS-T. And allowed to react at room temperature for 1 hour. Thereafter, the solution was discarded and washed, and then 100 μL of a horseradish peroxidase (HRP) -labeled streptavidin (Jackson) solution was added to 1 well and allowed to react at room temperature for 1 hour. After discarding and washing the reaction solution, color development by 1StepUltra TMB substrate solution (Pierce) was measured by absorbance at 450 nm.
　その結果、（図１８）に示すように、多くの陽性クローンを確認出来た。また、これらでは陰性コントロールでは反応性が見られないことも合わせて確認された。モノクローナル抗体-ポリクローナル抗体ELISA系による反応性が確認されたが、他の組み合わせとしてポリクローナル抗体の他にモノクローナル抗体を用いても良い。その場合は、検出が可能な組み合わせにて行い、より感度の高い組み合わせを選択することが望ましい。 As a result, as shown in FIG. 18, many positive clones could be confirmed. It was also confirmed that no reactivity was seen in the negative control. Although the reactivity by the monoclonal antibody-polyclonal antibody ELISA system was confirmed, a monoclonal antibody other than the polyclonal antibody may be used as another combination. In that case, it is desirable to use combinations that allow detection, and to select combinations with higher sensitivity.
（実施例１０）本発明で得られた抗CSF1R抗体を用いた抗体-WFAレクチン サンドイッチELISA測定系
（１０－１）方法
　ここでは、抗CSF1Rモノクローナル抗体を使用して、該分子の抗体-WFAレクチン サンドイッチELISA測定系による検出を行った。抗CSF1Rモノクローナル抗体をそれぞれELISAプレート固相化側に使用し、一方WFAレクチンを検出側に用いた抗体-レクチンサンドイッチELISA測定系での使用の可否について検討した。抗体はELISAプレート固相化側でも検出側（液相側）でも、どちらに使用しても良く、もう一方の側にはレクチンを使用して（つまり抗体が固相側の場合はレクチンを液相側に使用して）、サンドイッチの検出系にて行うものである。一般的には感度が高く、バックグラウンドとなるノイズが少なくなる組み合わせにて検出系の構築を行う。また、WFAレクチンは市販のものを使用しても良いし、リコンビナントWFA、特にLDN特異的な単量体リコンビナントWFA(srWFA)を使用しても良い。
　各抗体をPBSで4μg/mLとなるように希釈し、ELISA用マイクロプレートに100uL/ウェルずつ添加した。4℃で一晩各抗体をプレートに吸着させた後、溶液を廃棄して、ウェルをPBS-T (PBS, 0.05％ Tween-20)洗浄した。次に、ブロッキング液(PBS with 3％ BSA)を300μL/ウェルで加えて、ブロッキングをした。前記ブロッキング液を廃棄し、洗浄した後、サンプル（CSF1Rリコンビナントタンパク質：R&D Recombinant Human M-CSFR Fc Chimera Cat#329-MR-100）の溶液100μLを各ウェルに添加した。37℃で2時間反応させた後、ウェル中の溶液を廃棄し、PBS-Tにて洗浄した後、ビオチン標識化したWFAレクチン（srWFA及びnWFA）をそれぞれ2μg/mLに調製して、室温で1時間反応させた。その後、溶液を廃棄して洗浄後、西洋ワサビペルオキシダーゼ（HRP）標識ストレプトアビジン（Jackson社）溶液を1ウェルに100μL加えて1時間室温にて反応させた。反応液を廃棄、洗浄した後、1StepUltra TMB基質液（Pierce社）による発色を450nmの吸光度で測定した。 (Example 10) Antibody-WFA lectin using anti-CSF1R antibody obtained in the present invention Sandwich ELISA measurement system (10-1) method Here, anti-CSF1R monoclonal antibody is used and antibody-WFA lectin of the molecule is used. Detection was performed by a sandwich ELISA measurement system. The anti-CSF1R monoclonal antibody was used on the ELISA plate immobilization side, while the feasibility of use in an antibody-lectin sandwich ELISA measurement system using WFA lectin on the detection side was examined. The antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. Used on the phase side) in a sandwich detection system. In general, a detection system is constructed with a combination of high sensitivity and low background noise. In addition, commercially available WFA lectins may be used, or recombinant WFA, particularly LDN-specific monomeric recombinant WFA (srWFA) may be used.
Each antibody was diluted with PBS to 4 μg / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 μL / well for blocking. After the blocking solution was discarded and washed, 100 μL of a sample (CSF1R recombinant protein: R & D Recombinant Human M-CSFR Fc Chimera Cat # 329-MR-100) was added to each well. After reacting at 37 ° C. for 2 hours, the solution in the well was discarded and washed with PBS-T. Then, biotin-labeled WFA lectins (srWFA and nWFA) were each adjusted to 2 μg / mL, Reacted for 1 hour. Thereafter, the solution was discarded and washed, and then 100 μL of a horseradish peroxidase (HRP) -labeled streptavidin (Jackson) solution was added to 1 well and allowed to react at room temperature for 1 hour. After discarding and washing the reaction solution, color development by 1StepUltra TMB substrate solution (Pierce) was measured by absorbance at 450 nm.
（１０－２）結果
　抗CSF1Rモノクローナル抗体を固相側にし、検出側にWFAレクチンを使用した検出系での結果について、代表例としてCSR-3、CSR-4、CSR-18、CSR-21、CSR-30クローンの抗体について（図１９、上段はsrWFA，下段はnWFA）に示す。逆に、WFAレクチンを固相側にし、検出側に抗CSF1Rモノクローナル抗体を使用した検出系での結果について、代表例としてCSR-3、CSR-4、CSR-18、CSR-21、CSR-30クローンの抗体について（図２０、上段はsrWFA，下段はnWFA ）に示す。これらの結果から多くのクローンの抗体について、抗原の濃度依存的に反応性が認められた。 (10-2) Results As representative examples of the results of detection systems using anti-CSF1R monoclonal antibody on the solid phase side and WFA lectin on the detection side, CSR-3, CSR-4, CSR-18, CSR-21, The CSR-30 clone antibody is shown in FIG. 19, srWFA on the top and nWFA on the bottom. Conversely, as a representative example of the detection results using WFA lectin on the solid phase side and anti-CSF1R monoclonal antibody on the detection side, CSR-3, CSR-4, CSR-18, CSR-21, CSR-30 The cloned antibody is shown in FIG. 20, srWFA on the top and nWFA on the bottom. From these results, reactivity of many clone antibodies was recognized depending on the concentration of antigen.
　図２１（左はsrWFA，右はnWFA）は、LDN糖鎖特異的単量体リコンビナントWFA(srWFA)又は市販の天然WFA(nWFA)と、抗CSF1R抗体とを組み合わせたサンドイッチELISA系を構築し、（実施例５）HEK293細胞で作製したLDN糖鎖を有するリコンビナントCSF1Rと、（実施例６）糖鎖遺伝子ノックアウト細胞で作製したLDN糖鎖を持っていないリコンビナントCSF1R、および市販CSF1R（R&D Recombinant Human M-CSFR Fc Chimera Cat#329-MR-100）に対する反応性を比較した。
　その結果、単量体リコンビナントWFAを用いた場合が、LDN糖鎖特異的にCSF1R分子を検出できていることが分かる。 FIG. 21 (left is srWFA, right is nWFA) is a sandwich ELISA system in which LDN sugar chain-specific monomeric recombinant WFA (srWFA) or commercially available natural WFA (nWFA) is combined with an anti-CSF1R antibody. (Example 5) Recombinant CSF1R having LDN sugar chains prepared in HEK293 cells, (Example 6) Recombinant CSF1R having no LDN sugar chains prepared in sugar chain gene knockout cells, and commercially available CSF1R (R & D Recombinant Human M -CSFR Fc Chimera Cat # 329-MR-100).
As a result, it can be seen that when the monomer recombinant WFA is used, the CSF1R molecule can be detected specifically in the LDN sugar chain.
（１０－３）抗CSF1Rモノクローナル抗体の評価
　各クローンが産生する抗CSF1R抗体について下記（表５）に結果をまとめた。 (10-3) Evaluation of anti-CSF1R monoclonal antibody The results of the anti-CSF1R antibody produced by each clone are summarized below (Table 5).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（実施例１１）抗体-WFAレクチン サンドイッチELISA測定系によるWFA + -CSF1R分子の検出
　（実施例８）で作成した抗CSF1R抗体を使用して、該分子の抗体-WFAレクチンサンドイッチELISA測定系による検出を行った。抗CSF1R抗体をそれぞれELISAプレート固相化側と検出側に用いサンドイッチELISA測定系の検討を行った。抗体はELISAプレート固相化側でも検出側（液相側）でも、どちらに使用しても良く、もう一方の側にはレクチンを使用して（つまり抗体が固相側の場合はレクチンを液相側に使用して）、サンドイッチの検出系にて行った。また、WFAレクチンとしては、市販の天然WFA及び単量体リコンビナントWFA（srWFA）を使用した。
　具体的には、各抗体をPBSで4μg/mLとなるように希釈し、ELISA用マイクロプレートに100uL/ウェルずつ添加した。4℃で一晩各抗体をプレートに吸着させた後、溶液を廃棄して、ウェルをPBS-T（PBS, 0.05％ Tween-20）洗浄した。次に、ブロッキング液（PBS with 3％ BSA）を300μL/ウェルで加えて、ブロッキングをした。前記ブロッキング液を廃棄し、洗浄した後、サンプル（実施例５）HEK293細胞で作製したLDN糖鎖を有するリコンビナントCSF1Rと、（実施例６）糖鎖遺伝子ノックアウト細胞で作製したLDN糖鎖を持っていないリコンビナントCSF1R、市販CSF1R（R&D Recombinant Human M-CSFR Fc Chimera Cat#329-MR-100）、および健常人血清プール（NHS））の溶液100μLを各ウェルに添加した。37℃で2時間反応させた後、ウェル中の溶液を廃棄し、PBS-Tにて洗浄した後、ビオチン標識化したWFAレクチン（あるいは単量体リコンビナントWFA： srWFA）をそれぞれ2μg/mLに調製して、室温で1時間反応させた．その後、溶液を廃棄して洗浄後、西洋ワサビペルオキシダーゼ（HRP）標識ストレプトアビジン（Jackson社）溶液を1ウェルに100μL加えて1時間室温にて反応させた。反応液を廃棄、洗浄した後、1StepUltra TMB基質液（Pierce社）による発色を450nmの吸光度で測定した。 (Example 11) Detection of WFA + -CSF1R molecule by antibody- WFA lectin sandwich ELISA measurement system (Example 8) and detection of the molecule by antibody- WFA lectin sandwich ELISA measurement system Went. The sandwich ELISA assay system was examined using anti-CSF1R antibodies on the ELISA plate immobilization side and the detection side, respectively. The antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. (Used on the phase side) and performed in a sandwich detection system. As WFA lectins, commercially available natural WFA and monomeric recombinant WFA (srWFA) were used.
Specifically, each antibody was diluted with PBS to 4 μg / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 μL / well for blocking. After discarding and washing the blocking solution, the sample (Example 5) has a recombinant CSF1R having LDN sugar chains prepared in HEK293 cells, and (Example 6) has an LDN sugar chain prepared in sugar chain gene knockout cells. 100 μL of a solution of no recombinant CSF1R, commercially available CSF1R (R & D Recombinant Human M-CSFR Fc Chimera Cat # 329-MR-100), and healthy human serum pool (NHS)) was added to each well. After reaction at 37 ° C for 2 hours, discard the solution in the well, wash with PBS-T, and then prepare biotin-labeled WFA lectin (or monomeric recombinant WFA: srWFA) to 2 µg / mL each. The reaction was allowed to proceed for 1 hour at room temperature. Thereafter, the solution was discarded and washed, and then 100 μL of a horseradish peroxidase (HRP) -labeled streptavidin (Jackson) solution was added to 1 well and allowed to react at room temperature for 1 hour. After discarding and washing the reaction solution, color development by 1StepUltra TMB substrate solution (Pierce) was measured by absorbance at 450 nm.
　その結果、（実施例８）で作成した抗CSF1R抗体CSR-3、CSR-4、CSR-18、CSR-21、CSR-30は、単量体srWFA、nWFAのいずれと組み合わせても、CSF1R分子上に存在するWFA/VVA結合性糖鎖を検出できることが示された（図２２）。特に、LDN特異的な単量体srWFAはLDN糖鎖の有無を特異的に識別できることが示された（図２２上段）。なお、nWFAでは僅かにLDN糖鎖を持たないrCSF1Rに対しても反応性が見られ、LDN以外の糖鎖にも反応していると考えられる。 As a result, the anti-CSF1R antibodies CSR-3, CSR-4, CSR-18, CSR-21, and CSR-30 prepared in (Example 8) can be combined with either monomeric srWFA or nWFA. It was shown that the WFA / VVA-binding sugar chain present above can be detected (FIG. 22). In particular, it was shown that the LDN-specific monomer srWFA can specifically identify the presence or absence of LDN sugar chains (upper part of FIG. 22). It should be noted that nWFA has a reactivity with rCSF1R that does not have a LDN sugar chain slightly, and is considered to react with sugar chains other than LDN.
（実施例１２）抗体CRS-3を用いた抗体-WFAレクチンサンドイッチ、および抗体-抗体サンドイッチELISA測定系によるCSF1R分子の検出
　（実施例８）で作製した抗CSF1R抗体（CSR-3）、WFAレクチン、および市販抗体（R&D Systems）を使用して、CSR-3-WFAレクチンサンドイッチELISAとCSR-3-市販抗体サンドイッチELISA測定系により、同じ濃度（の希釈系列）に調整したrCSF1R(LDN+)およびrCSF1R(LDN-)の検出を行った。
　具体的には、各抗体をPBSで4μg/mLとなるように希釈し、ELISA用マイクロプレートに100uL/ウェルずつ添加した。4℃で一晩各抗体をプレートに吸着させた後、溶液を廃棄して、ウェルをPBS-T (PBS, 0.05％ Tween-20)洗浄した。次に、ブロッキング液(PBS with 3％ BSA)を300μL/ウェルで加えて、ブロッキングをした。前記ブロッキング液を廃棄し、洗浄した後、同じCSF1R濃度（の希釈系列）になるように調整した（実施例５）HEK293細胞で作製したLDN糖鎖を有するリコンビナントCSF1Rと（実施例６）糖鎖遺伝子ノックアウト細胞で作製したLDN糖鎖を持っていないリコンビナントCSF1R、および健常人血清プール（NHS））の溶液100μLを各ウェルに添加した。37℃で2時間反応させた後、ウェル中の溶液を廃棄し、PBS-Tにて洗浄した後、ビオチン標識化したWFAレクチン（あるいは単量体リコンビナントWFA： srWFA）あるいは市販ビオチン標識抗CSF1R抗体（R&D biotinylated anti-CSF1R pAb Cat#BAF329）をそれぞれ2μg/mLに調製して、室温で1時間反応させた．その後、溶液を廃棄して洗浄後、西洋ワサビペルオキシダーゼ（HRP）標識ストレプトアビジン（Jackson社）溶液を1ウェルに100μL加えて1時間室温にて反応させた。反応液を廃棄、洗浄した後、1StepUltra TMB基質液（Pierce社）による発色を450nmの吸光度で測定した。 (Example 12) Antibody-WFA lectin sandwich using antibody CRS-3, and anti-CSF1R antibody (CSR-3) and WFA lectin prepared by detection of CSF1R molecule by antibody-antibody sandwich ELISA measurement system (Example 8) , And rCSF1R (LDN +) and rCSF1R adjusted to the same concentration (by dilution series) using CSR-3-WFA lectin sandwich ELISA and CSR-3-commercial antibody sandwich ELISA measurement system using commercially available antibodies (R & D Systems) (LDN-) was detected.
Specifically, each antibody was diluted with PBS to 4 μg / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 μL / well for blocking. The blocking solution was discarded and washed, and then adjusted to have the same CSF1R concentration (dilution series thereof) (Example 5) Recombinant CSF1R having LDN sugar chains prepared in HEK293 cells and (Example 6) sugar chains 100 μL of a recombinant CSF1R without a LDN sugar chain prepared from gene knockout cells and a healthy human serum pool (NHS) solution were added to each well. After reacting at 37 ° C for 2 hours, discard the solution in the well, wash with PBS-T, then biotin-labeled WFA lectin (or monomeric recombinant WFA: srWFA) or commercially available biotin-labeled anti-CSF1R antibody (R & D biotinylated anti-CSF1R pAb Cat # BAF329) was prepared at 2 μg / mL and allowed to react at room temperature for 1 hour. Thereafter, the solution was discarded and washed, and then 100 μL of a horseradish peroxidase (HRP) -labeled streptavidin (Jackson) solution was added to 1 well and allowed to react at room temperature for 1 hour. After discarding and washing the reaction solution, color development by 1StepUltra TMB substrate solution (Pierce) was measured by absorbance at 450 nm.
　その結果、（実施例８）で作成した抗CSF1R抗体（CSR-3）は、CSF1R (LDN+)およびrCSF1R (LDN-)のいずれも、ほぼ同じように検出できることを確認した（図２３下段）。一方WFAレクチンで検出した場合はCSF1R(LDN+)を感度良く検出し、CSF1R(LDN-)の反応性は低い（図２３上段）。また、srWFAを使用したときに、よりLDN糖鎖を含有するCSF1R特異的に検出が出来ていることが確認された。 As a result, it was confirmed that the anti-CSF1R antibody (CSR-3) prepared in (Example 8) was able to detect both CSF1R (LDN +) and rCSF1R (LDN-) in almost the same manner (lower part of Fig. 23). On the other hand, when detected with WFA lectin, CSF1R (LDN +) is detected with high sensitivity, and the reactivity of CSF1R (LDN-) is low (upper part of FIG. 23). Moreover, when srWFA was used, it was confirmed that the detection was more specific to CSF1R containing LDN sugar chains.
（実施例１３）抗体-VVAレクチンサンドイッチELISA測定系によるマーカー分子の検出
　WFAレクチンの代わりに、VVAレクチンなどのLacdiNAc / GalNAc結合性レクチンを使用することも出来る。そこで、抗CSF1R抗体を使用して、該分子の抗体-VVAレクチンサンドイッチELISA測定系による検出を行った。抗CSF1R抗体をそれぞれELISAプレート固相化側と検出側に用いサンドイッチELISA測定系の検討を行った。抗体はELISAプレート固相化側でも検出側（液相側）でも、どちらに使用しても良く、もう一方の側にはレクチンを使用して（つまり抗体が固相側の場合はレクチンを液相側に使用して）、サンドイッチの検出系にて行うものである。一般的には感度が高く、バックグラウンドとなるノイズが少なくなる組み合わせにて検出系の構築を行う。
　各抗体をPBSで4μg/mLとなるように希釈し、ELISA用マイクロプレートに100uL/ウェルずつ添加した。4℃で一晩各抗体をプレートに吸着させた後、溶液を廃棄して、ウェルをPBS-T (PBS, 0.05％ Tween-20)洗浄した。次に、ブロッキング液（PBS with 3％ BSA）を300μL/ウェルで加えて、ブロッキングをした。前記ブロッキング液を廃棄し、洗浄した後、サンプル（実施例５）HEK293細胞で作製したLDN糖鎖を有するリコンビナントCSF1Rと、（実施例６）糖鎖遺伝子ノックアウト細胞で作製したLDN糖鎖を持っていないリコンビナントCSF1Rおよび健常人血清プール（NHS））の溶液100μLを各ウェルに添加した。37℃で2時間反応させた後、ウェル中の溶液を廃棄し、PBS-Tにて洗浄した後、ビオチン標識VVAレクチン（Vector Laboratories）を2μg/mLに調製して、室温で1時間反応させた．その後、溶液を廃棄して洗浄後、西洋ワサビペルオキシダーゼ（HRP）標識ストレプトアビジン（Jackson社）溶液を1ウェルに100μL加えて1時間室温にて反応させた。反応液を廃棄、洗浄した後、1StepUltra TMB基質液（Pierce社）による発色を450nmの吸光度で測定した。
抗CSF1R抗体（CSR-3）を固相側に用いた結果について（図２４）に示す。この結果から、VVAレクチンを用いた検出系によってもWFA+-CSF1Rを検出・測定することが出来ることが明らかとなった。 (Example 13) Detection of marker molecule by antibody-VVA lectin sandwich ELISA measurement system In place of WFA lectin, LacdiNAc / GalNAc binding lectin such as VVA lectin can also be used. Therefore, the anti-CSF1R antibody was used to detect the molecule by an antibody-VVA lectin sandwich ELISA measurement system. The sandwich ELISA assay system was examined using anti-CSF1R antibodies on the ELISA plate immobilization side and the detection side, respectively. The antibody may be used on either the ELISA plate immobilization side or the detection side (liquid phase side), and lectin is used on the other side. Used on the phase side) in a sandwich detection system. In general, a detection system is constructed with a combination of high sensitivity and low background noise.
Each antibody was diluted with PBS to 4 μg / mL and added to an ELISA microplate at 100 uL / well. After each antibody was adsorbed to the plate at 4 ° C. overnight, the solution was discarded and the wells were washed with PBS-T (PBS, 0.05% Tween-20). Next, blocking solution (PBS with 3% BSA) was added at 300 μL / well for blocking. After discarding and washing the blocking solution, the sample (Example 5) has a recombinant CSF1R having LDN sugar chains prepared in HEK293 cells, and (Example 6) has an LDN sugar chain prepared in sugar chain gene knockout cells. 100 μL of a solution of no recombinant CSF1R and healthy human serum pool (NHS) was added to each well. After reacting at 37 ° C for 2 hours, discard the solution in the well and wash with PBS-T. Then, prepare biotin-labeled VVA lectin (Vector Laboratories) at 2 μg / mL and react at room temperature for 1 hour. It was. Thereafter, the solution was discarded and washed, and then 100 μL of a horseradish peroxidase (HRP) -labeled streptavidin (Jackson) solution was added to 1 well and allowed to react at room temperature for 1 hour. After discarding and washing the reaction solution, color development by 1StepUltra TMB substrate solution (Pierce) was measured by absorbance at 450 nm.
The results of using anti-CSF1R antibody (CSR-3) on the solid phase side are shown in FIG. From this result, it became clear that WFA + -CSF1R can also be detected and measured by a detection system using VVA lectin.
（実施例１４）本発明の抗CSF1R抗体によるWFA + -CSF1Rの検出
（１４－１）WFAレクチン－抗CSF1R抗体サンドイッチELISA系による検出
　本実施例では、（実施例８）で作製した抗CSF1Rモノクローナル抗体が肝疾患マーカー分子WFA+-CSF1Rの検出に使用可能であることを確認するために、非特許文献２に記載の方法に従って、抗CSF1R抗体-WFAレクチンサンドイッチELISA法に適用し、血清中のWFA＋-CSF1R値を測定した。
　具体的には、被検体血清として、健常人血清プール血清（17名分、NHS）、HBV感染肝細胞がん患者プール血清（K1）、HCV感染肝がん患者プール血清（K2）、HCV感染肝細胞がん患者（脾摘済み）プール血清（K3）、を使用した。（実施例８）で作製した抗CSF1R抗体のうちCSR-3、CSR-4、CSR-18、CSR-21、CSR-30を用い、コートしたMaxisorp（登録商標）イムノプレ－ト上に血清試料（遮断緩衝液で1:20に希釈）を37℃にて2時間作用させた。その後、緩衝液で10分洗浄し、ビオチン化WFA（Vector Laboratories）を検出用プローブとして反応させた。次いで、洗浄後にHRP-結合ストレプトアビジンの1/50,000希釈液を100μ/wellで反応させた。反応後、緩衝液で6回洗浄し、基質溶液（100μL、Thermo Fisher Scientific社）を各ウェルに加え、適切な時間反応をさせた後、1M硫酸溶液で反応を終了させた。吸光度系にて450nm波長の吸光度を測定した。 (Example 14) Detection of WFA + -CSF1R with anti- CSF1R antibody of the present invention (14-1) Detection with WFA lectin-anti-CSF1R antibody sandwich ELISA system In this example, the anti-CSF1R monoclonal prepared in (Example 8) In order to confirm that the antibody can be used for detection of liver disease marker molecule WFA + -CSF1R, it was applied to anti-CSF1R antibody-WFA lectin sandwich ELISA according to the method described in Non-Patent Document 2 and WFA + -CSF1R values were measured.
Specifically, serum samples from healthy subjects (17 subjects, NHS), HBV-infected hepatocellular carcinoma patient pool serum (K1), HCV-infected liver cancer patient pool serum (K2), HCV infection Hepatocellular carcinoma patient (splenectomized) pooled serum (K3) was used. Among the anti-CSF1R antibodies prepared in Example 8, CSR-3, CSR-4, CSR-18, CSR-21, CSR-30 were used, and a serum sample (on a coated Maxisorp® immunoplate) ( (Diluted 1:20 with blocking buffer) at 37 ° C. for 2 hours. Then, it was washed with a buffer solution for 10 minutes and reacted with biotinylated WFA (Vector Laboratories) as a detection probe. Next, after washing, a 1 / 50,000 dilution of HRP-conjugated streptavidin was reacted at 100 μ / well. After the reaction, the plate was washed 6 times with a buffer solution, a substrate solution (100 μL, Thermo Fisher Scientific) was added to each well, and the reaction was allowed to proceed for an appropriate time, and then the reaction was terminated with a 1M sulfuric acid solution. Absorbance at a wavelength of 450 nm was measured with an absorbance system.
　その結果、抗体のクローン毎に差は認められるが、いずれの抗CSF1R抗体クローン（CSR-3、CSR-4、CSR-18、CSR-21、CSR-30）も健常人と比較して肝細胞がん患者でシグナルの増強が認められ、検査系に使用可能であることが確認された（図２５）。
　なお、（表５）に記載された他の抗CSF1R抗体（CSR-5、CSR-6など）もCSR-3、CSR-4、CSR-18の結果ほどの差ではないが、血清試料中の肝細胞がん検出用ELISAに用いることができることを示している（図２６）。 As a result, although there is a difference between antibody clones, all anti-CSF1R antibody clones (CSR-3, CSR-4, CSR-18, CSR-21, CSR-30) are hepatocytes compared to healthy individuals. Signal enhancement was observed in cancer patients, confirming that it could be used in a test system (FIG. 25).
In addition, other anti-CSF1R antibodies (CSR-5, CSR-6, etc.) listed in (Table 5) are not as different as the results of CSR-3, CSR-4, CSR-18. This shows that it can be used for ELISA for detecting hepatocellular carcinoma (FIG. 26).
（１４－２）VVAレクチン－抗CSF1R抗体サンドイッチELISA系による検出
　本実験は、WFA に代えてVVAレクチンを用いても、抗CSF1R抗体とのサンドイッチELISAで、WFAと同様に肝細胞がん患者由来pool血清と健常人由来のpool血清とを識別できることを確認するための実験である。
　具体的には、（１４－１）と同様に、健常人血清プール血清（17名分、NHS）、HBV感染肝細胞がん患者プール血清（K1）、HCV感染肝がん患者プール血清（K2）、HCV感染肝細胞がん患者（脾摘済み）プール血清（K3）を被検体血清とし、（実施例８）で作製した抗CSF1R抗体のうちCSR-18を用いて、（１４－１）の方法にならい、ビオチン化WFA（Vector Laboratories）及びビオチン化VVA（Vector Laboratories）を検出用プローブとして反応させた。
　次いで、洗浄後にHRP-結合ストレプトアビジンの1/20,000希釈液を100μ/wellで反応させた。反応後、緩衝液で4回洗浄し、基質溶液（100μL、Thermo Fisher Scientific社）を各ウェルに加え、適切な時間反応をさせた後、1M硫酸溶液で反応を終了させた。吸光度系にて450nm波長の吸光度を測定した。
　その結果、抗体CSR-18とVVAレクチンとのサンドイッチELISA測定系においても、WFAレクチンとのサンドイッチELISA測定系の場合と同様に、健常人と比較して肝細胞がん患者でシグナルの増強が認められ、肝細胞がん患者血清pool血清と健常人のpool血清とを識別できることが確認された（図２７）。
　このことは、VVAレクチンが、実際の臨床体液試料を用いた場合でも、WFAレクチンと遜色なく、肝細胞がん患者で特異的に増大するCSF1R上の糖鎖に対する結合性を発揮すること、すなわちVVAレクチンを抗CSF1R抗体と組み合わせたアッセイ系に使用することで、臨床体液試料中の「WFA／VVAレクチン結合性糖鎖含有CSF1R量」を測定可能であることを意味する。 (14-2) Detection by VVA lectin-anti-CSF1R antibody sandwich ELISA system In this experiment, even if VVA lectin was used instead of WFA, it was a sandwich ELISA with anti-CSF1R antibody and was derived from a hepatocellular carcinoma patient as in WFA. This is an experiment to confirm that pool serum can be distinguished from pool serum derived from healthy individuals.
Specifically, as in (14-1), healthy human serum pool serum (for 17 patients, NHS), HBV-infected hepatocellular carcinoma patient pool serum (K1), HCV-infected liver cancer patient pool serum (K2 ), HCV-infected hepatocellular carcinoma patient (splenectomized) pooled serum (K3) as the subject serum, and using CSR-18 among the anti-CSF1R antibodies prepared in (Example 8), (14-1) According to the method, biotinylated WFA (Vector Laboratories) and biotinylated VVA (Vector Laboratories) were reacted as detection probes.
Next, after washing, a 1 / 20,000 dilution of HRP-conjugated streptavidin was reacted at 100 μ / well. After the reaction, the plate was washed 4 times with a buffer solution, a substrate solution (100 μL, Thermo Fisher Scientific) was added to each well, and the reaction was allowed to proceed for an appropriate time, and then the reaction was terminated with a 1M sulfuric acid solution. Absorbance at a wavelength of 450 nm was measured with an absorbance system.
As a result, in the sandwich ELISA assay system for antibody CSR-18 and VVA lectin, as in the sandwich ELISA assay system for WFA lectin, signal enhancement was observed in hepatocellular carcinoma patients compared to healthy individuals. It was confirmed that the serum pool serum of hepatocellular carcinoma patients can be distinguished from the pool serum of healthy individuals (FIG. 27).
This means that VVA lectin exhibits a binding ability to glycans on CSF1R that is specifically increased in patients with hepatocellular carcinoma, inferior to WFA lectin even when using actual clinical body fluid samples. By using VVA lectin in an assay system in combination with an anti-CSF1R antibody, it means that “the amount of CSF1R containing a WFA / VVA lectin-binding sugar chain” in a clinical body fluid sample can be measured.
（実施例１５）「全CSF1R量」測定用のELISAとして用いることのできる「CSF1R特異的糖鎖結合性レクチン-抗CSF1R抗体サンドイッチELISA」系の構築
　本実施例では、本発明の肝細胞がん発症リスク指数である「WFA+-CSF1R％」の測定、すなわち被検試料中の「全CSF1R量」に対する「WFA／VVA結合性糖鎖を有するCSF1R量」の比率は、「CSF1R特異的レクチン結合性糖鎖を有するCSF1R量」に対する「WFA／VVA結合性糖鎖を有するCSF1R量」を測定すれば良いことを実証する。
　具体的には、「CSF1R特異的レクチン」として典型的な「RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA」を用い、CSF1R特異的レクチン-抗CSF1R抗体サンドイッチ測定系を構築し、当該レクチン-抗体サンドイッチアッセイ系が全CSF1R測定のための2つの抗CSF1R抗体を使用するサンドイッチ測定系に代え使用できることを実証する。
　なお、各レクチンによって糖鎖反応性（結合性）がそれぞれ異なるので、本実施例での測定値を比較する判定工程では、WFA（又はVVA）-CSF1R値との相対値での値（比率）として算出されたものを使用した。 (Example 15) Construction of "CSF1R-specific glycan-binding lectin-anti-CSF1R antibody sandwich ELISA" system that can be used as an ELISA for measuring "total CSF1R amount" In this example, hepatocellular carcinoma of the present invention is used. The measurement of the onset risk index “WFA + -CSF1R%”, that is, the ratio of “CSF1R amount having WFA / VVA-binding sugar chain” to “total CSF1R amount” in the test sample is “CSF1R-specific lectin binding” It is demonstrated that “the amount of CSF1R having a WFA / VVA-binding sugar chain” relative to “the amount of CSF1R having a functional sugar chain” may be measured.
Specifically, CSF1R-specific lectin-anti-CSF1R antibody sandwich using typical `` RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, ConA '' as `` CSF1R-specific lectin '' A measurement system is constructed to demonstrate that the lectin-antibody sandwich assay system can be used in place of a sandwich measurement system that uses two anti-CSF1R antibodies for total CSF1R measurement.
In addition, since sugar chain reactivity (binding property) is different for each lectin, a value (ratio) relative to the WFA (or VVA) -CSF1R value in the determination step for comparing the measured values in this example. What was calculated as was used.
（１５－１）WFA+-CSF1Rおよび全CSF1R測定における希釈系の検討（BSA希釈液、10%NHS希釈液）
＜方法＞
　WFA+-CSF1Rおよび全CSF1Rは、本発明者らの既報（非特許文献２）の方法を一部改変して行った。Nunc Immobilizer Aminoプレート（サーモサイエンティフック、43613）を4μg/mL（実施例８）で作製した抗CSF1R抗体（CSR-3）で2時間コートし、洗浄バッファー（0.05% Tween20含有PBSバッファー, pH 7.4）で洗浄後、TBS（50mM Tris-pH8.0, 0.15M NaCl）により終夜4℃でブロッキングした。サンプルとして、（実施例５）の組換えCSF1R (LDN+)と（実施例６）の組換えCSF1R (LDN-)をそれぞれ疾患検体、正常検体由来CSF1Rの代替として用いた。希釈バッファーとして3%BSA, 0.1%Tween20含有PBSバッファー, pH7.4（BSA希釈液）または10%NHS含有の上記BSA希釈液（10%NHS希釈液）を用意し、上記組換えCSF1R(rCSF1R)の希釈溶液（1.11～810 ng/mlの範囲で複数段階を設定）を調製し、100μlをプレートにアプライして室温にて2時間振盪した。洗浄バッファーで4回洗浄後、検出用プローブとして、洗浄バッファーで希釈したビオチン結合の各レクチン（250～20,000倍希釈）[WFA（Vector Laboratory, B-1355, 5,000倍希釈）、VVA （Vector Laboratory, B-1235, 250倍希釈）、RCA120 （Vector Laboratory, B-1085, 20,000倍希釈）、DSA（Vector Laboratory, B-1185, 10,000倍希釈）、PHA-E4（J-オイルミルズ, J211, 2,000倍希釈）、SNA（Vector Laboratory, B-1305, 10,000倍希釈）、SSA（J-オイルミルズ, J218, 3,000倍希釈）、TJA-1（生化学工業, 300443, 500倍希釈）、LEL（Vector Laboratory, B-1175, 5,000倍希釈）、STL（Vector Laboratory, B-1165, 1,000倍希釈）、Con A（Vector Laboratory, B-1005, 20,000倍希釈）]、または抗CSF1R抗体（R&D Systems, BAF329、2,000倍希釈）を添加し室温にて１時間半振盪した。再び洗浄バッファーで４回洗浄後、20,000倍希釈HRP結合ストレプトアビジン溶液とともに１時間インキュベートし洗浄バッファーで４回洗浄した。各ウェルにTMB基質溶液（1-Step Ultra, サーモサイエンティフィック、34028）を加え反応を1M H2SO4で止めた後、450nmにおける吸光度を測定しELISAシグナル値とした。 (15-1) Examination of dilution system for BFA + -CSF1R and total CSF1R measurement (BSA dilution, 10% NHS dilution)
<Method>
WFA + -CSF1R and all CSF1R were obtained by a partial modification of the method previously reported by the present inventors (Non-patent Document 2). Nunc Immobilizer Amino plate (Thermo Scientific Hook, 43613) was coated with anti-CSF1R antibody (CSR-3) prepared at 4 μg / mL (Example 8) for 2 hours and washed buffer (PBS buffer containing 0.05% Tween20, pH 7.4) ) And then blocked with TBS (50 mM Tris-pH 8.0, 0.15 M NaCl) at 4 ° C. overnight. As samples, recombinant CSF1R (LDN +) of (Example 5) and recombinant CSF1R (LDN-) of (Example 6) were used as substitutes for CSF1R derived from diseased samples and normal samples, respectively. Prepare 3% BSA, 0.1% Tween20-containing PBS buffer, pH7.4 (BSA dilution) or 10% NHS-containing BSA dilution (10% NHS dilution) as the dilution buffer, and the above recombinant CSF1R (rCSF1R) A diluted solution (multiple steps were set in the range of 1.11 to 810 ng / ml) was prepared, and 100 μl was applied to the plate and shaken at room temperature for 2 hours. After washing 4 times with washing buffer, biotin-binding lectins diluted with washing buffer (250-20,000 times dilution) [WFA (Vector Laboratory, B-1355, 5,000 times dilution), VVA (Vector Laboratory, B-1235, diluted 250 times), RCA120 (Vector Laboratory, B-1085, diluted 20,000 times), DSA (Vector Laboratory, B-1185, diluted 10,000 times), PHA-E4 (J-Oil Mills, J211, 2,000 times diluted) Dilution), SNA (Vector Laboratory, B-1305, 10,000 times dilution), SSA (J-Oil Mills, J218, 3,000 times dilution), TJA-1 (Seikagaku, 300443, 500 times dilution), LEL (Vector Laboratory) , B-1175, 5,000-fold dilution), STL (Vector Laboratory, B-1165, 1,000-fold dilution), Con A (Vector Laboratory, B-1005, 20,000-fold dilution)], or anti-CSF1R antibody (R & D Systems, BAF329, (2,000-fold dilution) was added, and the mixture was shaken at room temperature for 1 hour and a half. After washing 4 times with the washing buffer again, it was incubated with a 20,000-fold diluted HRP-conjugated streptavidin solution for 1 hour and washed 4 times with the washing buffer. A TMB substrate solution (1-Step Ultra, Thermo Scientific, 34028) was added to each well, the reaction was stopped with 1M H 2 SO 4 , and the absorbance at 450 nm was measured to obtain an ELISA signal value.
＜結果＞
上記ELISAにおいて検出用プローブとして抗CSF1R抗体を用いた場合のシグナル値を全CSF1R測定値とし、組換えCSF1R [LacdiNAc(LDN)糖鎖(+)]と[LDN糖鎖(-)]の値が一致するよう濃度を調整した。濃度調整後の全CSF1R測定値はBSA希釈液、10%NHS希釈液のどちらを用いた場合もよく一致した（図２８Ａ，Ｂ）。このことはBSA希釈液系、10%NHS希釈液系のどちらでも、本測定系が問題無く使用可能であることを示している。 <Result>
The signal value when anti-CSF1R antibody is used as a detection probe in the above ELISA is the total CSF1R measurement value, and the values of recombinant CSF1R [LacdiNAc (LDN) sugar chain (+)] and [LDN sugar chain (-)] The concentration was adjusted to match. The total CSF1R measured values after concentration adjustment agreed well with both the BSA diluted solution and the 10% NHS diluted solution (FIGS. 28A and 28B). This indicates that this measurement system can be used without any problems in either the BSA dilution system or the 10% NHS dilution system.
（１５－２）全CSF1R値の代用としての各種CFS1R特異的レクチンの評価
　全CSF1R値の代用としての各種レクチンを評価するために、検出用プローブとしてビオチン標識各レクチンを用いた場合のシグナル値を濃度依存性のある範囲で平均値により標準化したものを相対的シグナル値として用いた。各レクチン-CSF1R相対的シグナル値に対するWFA-CSF1R相対的シグナル値の比を算出し、rCSF1R (LDN+)とrCSF1R (LDN-)それぞれ２点または３点のCSF1R濃度における平均値を、抗CSF1R抗体を用いた場合の全CFS1R値と比較した（図２９）。いずれのレクチンも抗CSF1R抗体を用いた場合と同様にrCSF1R (LDN+)の値がrCSF1R (LDN-)に対して高く、rCSF1R (LDN-)に対するrCSF1R (LDN+)の比は抗CSF1R抗体の場合が2.2であったのに対してレクチンの場合は2.0±0.25（1.7～2.6）であった。このことからRCA120、DSA、PHA-E4、SNA、SSA、TJA-1、LEL、STL、Con Aのいずれのレクチンも抗CSF1R抗体の代用として全CSF1R値の測定に用いることができる。以上より、全CSF1R値の代用として、「CSF1R特異的な共通糖鎖結合性レクチン」である各種レクチンの数値によって評価することが出来ると言える。また、疾患特異的なものとして、WFA-CSF1Rの代わりにVVA-CSF1Rも同様に測定、計算した結果も図２９に示している。 (15-2) Evaluation of various CFS1R-specific lectins as a substitute for the total CSF1R value In order to evaluate various lectins as a substitute for the total CSF1R value, the signal value when each biotin-labeled lectin was used as a detection probe. The relative signal value was normalized by the average value within a concentration-dependent range. Calculate the ratio of WFA-CSF1R relative signal value to each lectin-CSF1R relative signal value, rCSF1R (LDN +) and rCSF1R (LDN-), the mean value of CSF1R concentration at 2 or 3 points respectively, anti-CSF1R antibody Comparison was made with all CFS1R values when used (FIG. 29). As with any lectin, rCSF1R (LDN +) was higher than rCSF1R (LDN-), and the ratio of rCSF1R (LDN +) to rCSF1R (LDN-) was higher for anti-CSF1R antibodies. In the case of lectin, it was 2.0 ± 0.25 (1.7 to 2.6) compared to 2.2. Therefore, any of the lectins of RCA120, DSA, PHA-E4, SNA, SSA, TJA-1, LEL, STL, and Con A can be used to measure the total CSF1R value as a substitute for the anti-CSF1R antibody. From the above, it can be said that as a substitute for the total CSF1R value, it can be evaluated by the numerical values of various lectins which are “CSF1R-specific common sugar chain-binding lectins”. In addition, as a disease-specific one, VVA-CSF1R instead of WFA-CSF1R is also measured and calculated in the same manner as shown in FIG.
　また、レクチンはそれぞれが認識する糖鎖に対して異なる結合力を有するので、ELISAシグナル値（450nmにおける吸光度、図２９）はレクチンに依って強弱を認めている。そこで、測定対象CSF1R分子の濃度に補正して比較した。補正の方法は以下の通りである。
rCSF1R(LDN+)タンパク質は、前述の図１３にもある通り、LDN糖鎖を有しているが、そのLDN糖鎖の陽性率は全てのタンパク質のうちの約60％である。そこでrCSF1R(LDN+)濃度の補正を係数0.6として計算し補正値とした。このrCSF1R (LDN+)補正値を用いて標準曲線を作成し、これにrCSF1R (LDN-)シグナル値を当てはめて補正値（補正した相対濃度値）を算出し、相互に比較できるrCSF1R (LDN+)補正値、rCSF1R (LDN-)補正値として、これをグラフ化した（図３０）。この補正値での結果も同様に、いずれのレクチンも抗CSF1R抗体を用いた場合と同様にrCSF1R (LDN+)の値がrCSF1R (LDN-)に対して高く、WFAと各共通糖鎖プローブレクチンとの比率において、rCSF1R (LDN-)とrCSF1R (LDN+)に対する値の相対比は3.8±0.9（2.7～5.7）あった。図３０のように、平均値ではどのレクチンでもほぼ同じような量、傾向を示しており、このことからRCA120、DSA、PHA-E4、SNA、SSA、TJA-1、LEL、STL、Con Aのいずれのレクチンも抗CSF1R抗体の代用として全CSF1R値の測定に用いることができると考えられる。以上より、全CSF1R値の代用として、「CSF1R特異的な共通糖鎖結合性レクチン」である各種レクチンの数値によって評価することが出来ると言える。 Moreover, since each lectin has a different binding force to the sugar chain recognized by each lectin, the ELISA signal value (absorbance at 450 nm, FIG. 29) is observed to be strong or weak depending on the lectin. Therefore, the concentration of the CSF1R molecule to be measured was corrected and compared. The correction method is as follows.
The rCSF1R (LDN +) protein has an LDN sugar chain as shown in FIG. 13 described above, but the positive rate of the LDN sugar chain is about 60% of all proteins. Therefore, the correction of the rCSF1R (LDN +) concentration was calculated as a coefficient of 0.6 and used as a correction value. Using this rCSF1R (LDN +) correction value, create a standard curve, apply the rCSF1R (LDN-) signal value to this to calculate the correction value (corrected relative concentration value), and rCSF1R (LDN +) correction that can be compared with each other This was graphed as a value, rCSF1R (LDN-) correction value (FIG. 30). Similarly, the results with this correction value are similar to those obtained with the anti-CSF1R antibody for all lectins, and the value of rCSF1R (LDN +) is higher than that of rCSF1R (LDN-). The relative ratio of values to rCSF1R (LDN-) and rCSF1R (LDN +) was 3.8 ± 0.9 (2.7 to 5.7). As shown in Fig. 30, the average value shows almost the same amount and trend for any lectin. From this, RCA120, DSA, PHA-E4, SNA, SSA, TJA-1, LEL, STL, Con A Any lectin could be used to measure total CSF1R values as a substitute for anti-CSF1R antibodies. From the above, it can be said that as a substitute for the total CSF1R value, it can be evaluated by the numerical values of various lectins which are “CSF1R-specific common sugar chain-binding lectins”.
（１５－３）VVAを検出用プローブとした場合の希釈系の検討
　次に、（１５－１）に記載のサンプルを検出用プローブにVVAを用いて測定したところ、組換えCSF1R (LDN+)が濃度依存性にシグナル値が上昇したのに対して、(LDN-)ではシグナル値が殆ど上昇せず、またこの結果は10%NHS希釈液でも変わらなかったことから、VVAは疾患由来CSF1Rと正常CSF1Rを鑑別できるプローブであることが示された（図３１Ａ，Ｂ）。また、このことはバッファー（BSA希釈液）系、血清（10%NHS希釈液）系のどちらでも、本願の測定系が問題無く使用可能であることを示している。 (15-3) Examination of dilution system when VVA is used as detection probe Next, when the sample described in (15-1) was measured using VVA as a detection probe, recombinant CSF1R (LDN +) was found to be The signal value increased in a concentration-dependent manner, whereas the signal value did not increase significantly with (LDN-), and this result did not change even with 10% NHS dilution. It was shown to be a probe that can distinguish CSF1R (FIGS. 31A and 31B). This also indicates that the measurement system of the present application can be used without any problem in either a buffer (BSA dilution) system or a serum (10% NHS dilution) system.
（１５－４）抗体-各CSF1R特異的レクチン測定系における希釈系の検討
　また、同じ濃度（の希釈系列）に調整したrCSF1R (LDN+)およびrCSF1R (LDN-)を用いて、抗体-各CSF1R特異的共通糖鎖プローブレクチンでのサンドイッチ（ELISA）検出による全CSF1R測定を行った。ここでは、各共通糖鎖プローブレクチンとして幾つかをランダムに選択（LEL, STL, TJA-I）し、系の確認を行った。その結果、濃度調整後の全CSF1R測定値はBSA希釈液、10%NHS希釈液のどちらを用いた場合もよく一致していた（図３２Ａ～Ｆ）。このことはバッファー（BSA希釈液）系、血清（10%NHS希釈液）系のどちらでも、本願の測定系が問題無く使用可能であることを示している。 (15-4) Examination of dilution system in antibody-each CSF1R-specific lectin measurement system Also, using rCSF1R (LDN +) and rCSF1R (LDN-) adjusted to the same concentration (dilution series), antibody-specific each CSF1R Total CSF1R was measured by sandwich (ELISA) detection with a common sugar chain probe lectin. Here, some of the common sugar chain probe lectins were randomly selected (LEL, STL, TJA-I) to confirm the system. As a result, the total CSF1R measured values after concentration adjustment were in good agreement with both the BSA diluted solution and the 10% NHS diluted solution (FIGS. 32A to 32F). This indicates that the measurement system of the present application can be used without any problem in either a buffer (BSA dilution) system or a serum (10% NHS dilution) system.
（１５－５）
　以上より、前記（１５－１）～（１５－４）において構築された検出系（抗体-レクチン サンドイッチELISA系）が血清でも問題無く測定を行うことが出来るものであることが示唆された。
　また、これによって、例えばWFAやVVA などの疾患特異的プローブ（レクチン）とCSF1R共通糖鎖に結合性を有するプローブ（レクチン）を組み合わせたマルチレクチンアッセイによって、疾患特異的な糖鎖を有する分子量を測定することによって、その疾患に罹患しているのかどうかを判定することが可能であることが実証された。
　液体クロマトグラフィーや、抗体を用いた免疫沈降法・磁気ビーズ分離などの分離精製方法によってサンプルから分離精製されたターゲットとなる分子（CSF1Rタンパク質）の、複数のレクチンに対する結合量を測定することでも同様に疾患を判定することが出来る。このようなマルチレクチンアッセイによる測定方法は、臨床試料から分離精製されたCSF1Rタンパク質を1つないし2つ以上のレクチンでサンドイッチした複合体を形成させ、これを検出（定量）することで行うことが出来る。CSF1Rタンパク質とレクチンとの複合体の検出については、本願で行っているようなサンドイッチELISA系で検出することが出来る。本実施例で用いた「抗体-レクチンサンドイッチELISA」に代えて、疾患特異的プローブ（すなわちWFA又はVVAレクチン）またはCSF1R特異的共通糖鎖に結合性を有するプローブ（RCA120、DSA、PHA-E4、SNA、SSA、TJA-1、LEL、STL又はCon Aレクチン）のいずれか一方を固相化した後、臨床試料から分離精製されたCSF1Rタンパク質を加えて反応させ、さらにもう一方を液相側（検出側）にしてサンドイッチするようなELISA系として、（マルチ）レクチン-タンパク質複合体を形成させ、この複合体の量を検出してもよく、又はレクチン-レクチン サンドイッチ検出系による系でもよい。
　他にも、キャピラリー電気泳動法や、マイクロフリュイディクス技術を用いた分離・検出システムによって、CSF1Rタンパク質とレクチンとの複合体を検出することも出来る。さらには、酵素免疫測定法、二抗体サンドイッチELISA法、金コロイド法、放射免疫測定法、ラテックス凝集免疫測定法、蛍光免疫測定法、ウェスタンブロッティング法、免疫組織化学法、表面プラズモン共鳴法（SPR法）又は水晶振動子マイクロバランス（QCM）法等による定性的又は定量的手法などでも同様に測定が出来る。 (15-5)
From the above, it was suggested that the detection system (antibody-lectin sandwich ELISA system) constructed in the above (15-1) to (15-4) can be measured without problems even in serum.
In addition, the molecular weight of a disease-specific sugar chain can be determined by a multi-lectin assay that combines a disease-specific probe (lectin) such as WFA or VVA with a probe that binds to the CSF1R common sugar chain (lectin). It has been demonstrated that by measuring it is possible to determine whether or not the person is afflicted with the disease.
The same can be done by measuring the amount of target molecule (CSF1R protein) that is separated and purified from a sample by liquid chromatography, immunoprecipitation using an antibody, or separation and purification methods such as magnetic beads. Can determine the disease. Such a measurement method using a multi-lectin assay can be performed by forming a complex in which a CSF1R protein separated and purified from a clinical sample is sandwiched with one or more lectins, and detecting (quantifying) this complex. I can do it. About the detection of the complex of CSF1R protein and a lectin, it can detect with the sandwich ELISA system currently performed in this application. Instead of the “antibody-lectin sandwich ELISA” used in this example, a disease-specific probe (ie, WFA or VVA lectin) or a probe having binding ability to a CSF1R-specific common sugar chain (RCA120, DSA, PHA-E4, After solidifying any one of SNA, SSA, TJA-1, LEL, STL, or Con A lectin), the CSF1R protein separated and purified from the clinical sample is added and reacted, and the other is liquid side ( As an ELISA system for sandwiching on the detection side, a (multi) lectin-protein complex may be formed, and the amount of this complex may be detected, or a system using a lectin-lectin sandwich detection system may be used.
In addition, the complex of CSF1R protein and lectin can be detected by capillary electrophoresis or separation / detection system using microfluidics technology. Furthermore, enzyme immunoassay, two-antibody sandwich ELISA, gold colloid method, radioimmunoassay, latex agglutination immunoassay, fluorescence immunoassay, western blotting, immunohistochemistry, surface plasmon resonance (SPR method) ) Or a qualitative or quantitative method using a quartz crystal microbalance (QCM) method or the like.
１．マウス－マウスハイブリドーマ「CSR-3」
　受託番号：NITE BP-02117
　寄託日：2015年9月10日（2016年9月7日付で国際寄託に移管）
　寄託当局：独立行政法人製品評価技術基盤機構、特許微生物寄託センター(NPMD)
２．マウス－マウスハイブリドーマ「CSR-4」
　受託番号：NITE BP-02118
　寄託日：2015年9月10日（2016年9月7日付で国際寄託に移管）
　寄託当局：独立行政法人製品評価技術基盤機構、特許微生物寄託センター(NPMD)
３．マウス－マウスハイブリドーマ「CSR-18」
　受託番号：NITE BP-02119
　寄託日：2015年9月10日（2016年9月7日付で国際寄託に移管）
　寄託当局：独立行政法人製品評価技術基盤機構、特許微生物寄託センター(NPMD)
４．マウス－マウスハイブリドーマ「CSR-21」
　受託番号：NITE BP-02120
　寄託日：2015年9月10日（2016年9月7日付で国際寄託に移管）
　寄託当局：独立行政法人製品評価技術基盤機構、特許微生物寄託センター(NPMD)
５．マウス－マウスハイブリドーマ「CSR-30」
　受託番号：NITE BP-02121
　寄託日：2015年9月10日（2016年9月7日付で国際寄託に移管）
　寄託当局：独立行政法人製品評価技術基盤機構、特許微生物寄託センター(NPMD) 1. Mouse-mouse hybridoma "CSR-3"
Accession Number: NITE BP-02117
Deposit date: September 10, 2015 (transferred to international deposit on September 7, 2016)
Depositary authorities: National Institute for Product Evaluation Technology, Patent Microorganism Depositary Center (NPMD)
2. Mouse-mouse hybridoma "CSR-4"
Accession Number: NITE BP-02118
Deposit date: September 10, 2015 (transferred to international deposit on September 7, 2016)
Depositary authorities: National Institute for Product Evaluation Technology, Patent Microorganism Depositary Center (NPMD)
3. Mouse-mouse hybridoma "CSR-18"
Accession Number: NITE BP-02119
Deposit date: September 10, 2015 (transferred to international deposit on September 7, 2016)
Depositary authorities: National Institute for Product Evaluation Technology, Patent Microorganism Depositary Center (NPMD)
4). Mouse-mouse hybridoma "CSR-21"
Accession Number: NITE BP-02120
Deposit date: September 10, 2015 (transferred to international deposit on September 7, 2016)
Depositary authorities: National Institute for Product Evaluation Technology, Patent Microorganism Depositary Center (NPMD)
5). Mouse-mouse hybridoma "CSR-30"
Accession Number: NITE BP-02121
Deposit date: September 10, 2015 (transferred to international deposit on September 7, 2016)
Depositary authorities: National Institute for Product Evaluation Technology, Patent Microorganism Depositary Center (NPMD)
[規則26に基づく補充 12.10.2016]　
Figure WO-DOC-RO134-1

Figure WO-DOC-RO134-2
[Supplement under rule 26 12.10.2016]
Figure WO-DOC-RO134-1

Figure WO-DOC-RO134-2

Claims (32)

  1. 　肝硬変患者における肝細胞がんの発症リスク値の算出方法であって、（１）～（４）の工程を含む算出方法；
    （１）肝硬変患者の被験者から採取された一定容量の体液試料（以下、単に被検試料ともいう。）中の全CSF1R量（Ａ）を測定する工程、
    （２）被検試料中のWFA及び／又はVVA（以下、WFA／VVAと表記する。）結合性糖鎖含有CSF1R量（Ｂ）を測定する工程、及び
    （３）全CSF1Rに占めるWFA／VVA結合性糖鎖を含有するCSF1R量の比率（Ｃ）を、「Ｃ（％）＝（Ｂ）／（Ａ）×100」として算出する工程、
    （４）工程（３）で得られたＣ％の値を被験者の肝細胞がんの発症リスク値と決定する工程。     A method for calculating a risk of developing hepatocellular carcinoma in a cirrhotic patient, comprising the steps of (1) to (4);
    (1) a step of measuring the total amount of CSF1R (A) in a constant volume of body fluid sample (hereinafter also simply referred to as a test sample) collected from a subject of a cirrhotic patient;
    (2) a step of measuring WFA and / or VVA (hereinafter referred to as WFA / VVA) binding sugar chain-containing CSF1R amount (B) in a test sample, and (3) WFA / VVA in the total CSF1R Calculating the ratio (C) of the amount of CSF1R containing a binding sugar chain as “C (%) = (B) / (A) × 100”;
    (4) A step of determining the value of C% obtained in step (3) as the risk of developing hepatocellular carcinoma of the subject.
  2. 　（１）の全CSF1R量を測定する工程が、少なくとも2種類の抗CSF1R抗体を用いるサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、精製CSF1R量を測定することを特徴とする、請求項１に記載の方法。 The step of measuring the total amount of CSF1R in (1) is measured by a sandwich assay system using at least two types of anti-CSF1R antibodies, or CSF1R is purified from a test sample using anti-CSF1R antibodies, and the amount of purified CSF1R is determined. The method according to claim 1, wherein the measurement is performed.
  3. 　（１）の被検試料中の全CSF1R量（Ａ）を測定する工程が、被検試料中のCSF1R特異的レクチン結合性糖鎖含有CSF1R量を測定するものであり、少なくともCSF1R特異的レクチンと、抗CSF1R抗体とを含むサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、CSF1R特異的レクチンと結合する精製CSF1R量を測定することを特徴とする、請求項１に記載の方法。 The step (1) of measuring the total CSF1R amount (A) in the test sample is a method for measuring the CSF1R-specific lectin-binding sugar chain-containing CSF1R amount in the test sample, and at least a CSF1R-specific lectin and Measuring CSF1R from a test sample using an anti-CSF1R antibody and measuring the amount of purified CSF1R that binds to a CSF1R-specific lectin. The method of claim 1.
  4. 　CSF1R特異的レクチンが、RCA120, DSA, PHA-E4、SNA、SSA、TJA-I、LEL、STL及びConAからなる群から選択される少なくとも１つのレクチンである、請求項３に記載の方法。 The method according to claim 3, wherein the CSF1R-specific lectin is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA.
  5. 　（２）のWFA／VVA結合性糖鎖含有CSF1R量（Ｂ）を測定する工程が、少なくともWFA／VVAレクチンと、抗CSF1R抗体とを含むサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、LDN特異的レクチンと結合する精製CSF1R量を測定することを特徴とする、請求項１～４のいずれか一項に記載の方法。 The step (2) of measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain (B) is measured by a sandwich assay system containing at least a WFA / VVA lectin and an anti-CSF1R antibody, or anti-CSF1R from a test sample. The method according to any one of claims 1 to 4, wherein CSF1R is purified using a CSF1R antibody, and the amount of purified CSF1R that binds to an LDN-specific lectin is measured.
  6. 　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAのいずれかのレクチンから選択される少なくとも１つのレクチンである、請求項５に記載の方法。 The method according to claim 5, wherein the WFA / VVA lectin is at least one lectin selected from any one of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
  7. 　工程（１）及び工程（２）が、少なくともCSF1R特異的レクチン及びWFA／VVAレクチンと抗CSF1R抗体とを用いて同時に行われる工程であって、両レクチン及び抗CSF1R抗体とを含む同一のサンドイッチアッセイ系を用いて測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製した後、各レクチンと結合するCSF1R量を同一のアッセイ系で測定することを特徴とする、請求項３～６のいずれか一項に記載の方法。 Step (1) and Step (2) are performed simultaneously using at least a CSF1R-specific lectin and a WFA / VVA lectin and an anti-CSF1R antibody, and include both lectins and an anti-CSF1R antibody. The CSF1R that binds to each lectin is measured in the same assay system after measurement using a system or after purification of CSF1R from a test sample using an anti-CSF1R antibody. 7. The method according to any one of 6.
  8. 　肝硬変患者における肝細胞がんの発症リスク値を判定する方法であって、（１）～（３）の工程を含む方法；
    （１）肝硬変患者である被験者の肝細胞がんの発症リスク値（Ｃ％）を請求項１～７のいずれか一項に記載の方法に従い算出する工程、
    （２）あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者から採取された各体液試料に対し、工程（１）と同じ算出工程により、各々の全CSF1Rに占めるWFA／VVA結合性糖鎖を含有するCSF1R量の比率（Ｃｎ）と、各々の患者をフォローアップして得た肝細胞がん発症率データと対比させて肝細胞がん発症の最適カットオフ値（Ｍ％）を算出する工程、
    （３）工程（１）で算出された肝細胞がんの発症リスク値（Ｃ％）を（２）で算出された最適カットオフ値（Ｍ％）と比較して上回っている場合に被験者の肝細胞がん発症リスクが有意に高いと判定し、最適カットオフ値未満であれば発症リスクが有意に低いと判定する方法。     A method for determining the risk of developing hepatocellular carcinoma in a cirrhotic patient, comprising the steps of (1) to (3);
    (1) a step of calculating a risk of developing hepatocellular carcinoma (C%) of a subject who is a cirrhotic patient according to the method according to any one of claims 1 to 7,
    (2) A WFA / VVA-binding glycan occupying in each CSF1R is calculated for each body fluid sample collected from a liver parameter cirrhosis patient who does not have hepatocellular carcinoma in advance by the same calculation step as in step (1). The optimal cut-off value (M%) for the development of hepatocellular carcinoma is calculated by comparing the ratio (Cn) of the amount of CSF1R containing HC1 and the hepatocellular carcinoma incidence data obtained by following up each patient Process,
    (3) If the risk of developing hepatocellular carcinoma (C%) calculated in step (1) is higher than the optimal cutoff value (M%) calculated in (2), A method for determining that the risk of developing hepatocellular carcinoma is significantly high, and determining that the risk of developing is significantly low if the risk is less than the optimum cutoff value.
  9. 　前記最適カットオフ値が35.0±10.0％の値である請求項８に記載の方法。 The method according to claim 8, wherein the optimum cutoff value is a value of 35.0 ± 10.0%.
  10. 　肝硬変患者における予後判定指数値の算出方法であって、（１）及び（２）の工程を含む方法；
    （１）肝硬変患者である被験者から採取された一定容量の体液試料（被検試料）中のWFA／VVA結合性糖鎖を含有するCSF1R量（Ｂ）を測定する工程、
    （２）工程（１）で得られたＢng/mlの値を、被験者の予後判定指数値と決定する工程。     A method for calculating a prognostic index value in a patient with cirrhosis, comprising the steps of (1) and (2);
    (1) a step of measuring the amount of CSF1R (B) containing a WFA / VVA-binding sugar chain in a constant volume of body fluid sample (test sample) collected from a subject who has cirrhosis;
    (2) A step of determining the value of Bng / ml obtained in step (1) as the prognosis determination index value of the subject.
  11. 　（１）のWFA／VVA結合性糖鎖含有CSF1R量（Ｂ）を測定する工程が、少なくともWFA／VVAレクチンと、抗CSF1R抗体とを含むサンドイッチアッセイ系により測定するか、又は被検試料から抗CSF1R抗体を用いてCSF1Rを精製し、LDN特異的レクチンと結合する精製CSF1R量を測定することを特徴とする、請求項１０に記載の方法。 The step (1) of measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain (B) is measured by a sandwich assay system containing at least a WFA / VVA lectin and an anti-CSF1R antibody, or anti-CSF1R from a test sample. The method according to claim 10, wherein CSF1R is purified using a CSF1R antibody, and the amount of purified CSF1R that binds to an LDN-specific lectin is measured.
  12. 　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAのいずれかのレクチンから選択される少なくとも１つのレクチンである、請求項１１に記載の方法。 The method according to claim 11, wherein the WFA / VVA lectin is at least one lectin selected from any one of a natural WFA, a recombinant WFA, a monomeric recombinant WFA, and a VVA.
  13. 　肝硬変患者における予後を判定する方法であって、（１）～（３）の工程を含む方法；
    （１）肝硬変患者である被験者の予後判定指数値（Ｂng/ml）を請求項１０～１２のいずれか一項に記載の方法に従い算出する工程、
    （２）あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者から採取された各体液試料に対し、工程（１）と同じ算出工程により、各々のWFA／VVA結合性糖鎖を含有するCSF1R量（Ｂｎ）と、各々の患者をフォローアップして得た累積生存率データと対比させて肝細胞がん患者予後の最適カットオフ値（Ｎng/ml）を算出する工程、
    （３）工程（１）で算出された予後判定指数値（Ｂng/ml）を（２）で算出された最適カットオフ値（Ｎng/ml）と比較して上回っている場合に被験者の被験者の予後が有意に悪いと判定し、最適カットオフ値未満であれば被験者の予後が有意に良いと判定する方法。     A method for determining the prognosis in a cirrhotic patient, comprising the steps of (1) to (3);
    (1) a step of calculating a prognosis index value (Bng / ml) of a subject who is a cirrhosis patient according to the method according to any one of claims 10 to 12,
    (2) CSF1R containing each WFA / VVA-binding glycan is obtained by the same calculation process as in step (1) on each body fluid sample collected from a liver cirrhosis patient who does not have hepatocellular carcinoma in advance. Calculating the optimal cutoff value (Nng / ml) of hepatocellular carcinoma patient prognosis by comparing the amount (Bn) and cumulative survival data obtained by following up each patient;
    (3) When the prognosis determination index value (Bng / ml) calculated in step (1) is higher than the optimal cutoff value (Nng / ml) calculated in (2), A method of determining that the prognosis is significantly poor and that the prognosis of the subject is significantly good if it is less than the optimum cutoff value.
  14. 　前記最適カットオフ値が310±100ng/mlの値である請求項１３に記載の方法。 The method according to claim 13, wherein the optimum cutoff value is a value of 310 ± 100 ng / ml.
  15. 　WFA／VVA結合性糖鎖含有CSF1Rを検出又は定量するためのレクチン－抗体サンドイッチアッセイであって、
    WFA／VVAレクチンと、
    CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体とを用い、（１）～（３）の工程を含むアッセイ；
    （１）液相中で前記レクチン又は前記抗CSF1R抗体のいずれかと被検試料を接触させ、被検試料中のCSF1Rとの複合体を形成させる工程、
    （２）（１）で得られたレクチン又は抗体とのCSF1R複合体を、分離し、又は分離せずに他方が溶解もしくは分散されている検出用液相中で、他方とCSF1R複合体を結合させ、レクチン及び抗体でサンドイッチされたCSF1R複合体を得る工程、
    （３）（２）で得られたレクチン及び抗体サンドイッチCSF1R複合体量を検出又は定量する工程。     A lectin-antibody sandwich assay for detecting or quantifying CSF1R containing a WFA / VVA-binding sugar chain comprising:
    WFA / VVA lectin,
    CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13, CSR- An assay comprising the steps of (1) to (3) using at least one anti-CSF1R antibody selected from the group consisting of 26, CSR-27, and CSR-29 antibodies;
    (1) contacting the test sample with either the lectin or the anti-CSF1R antibody in a liquid phase to form a complex with CSF1R in the test sample;
    (2) The CSF1R complex with the lectin or antibody obtained in (1) is separated, or the other is bound to the CSF1R complex in the detection liquid phase in which the other is dissolved or dispersed. Obtaining a CSF1R complex sandwiched with a lectin and an antibody,
    (3) A step of detecting or quantifying the amount of the lectin and antibody sandwich CSF1R complex obtained in (2).
  16. 　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンである、請求項１５に記載のアッセイ。 The assay according to claim 15, wherein the WFA / VVA lectin is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
  17. 　WFA／VVA結合性糖鎖含有CSF1Rを検出又は定量するためのレクチン－抗体サンドイッチアッセイ用キットであって、（１）及び（２）を含む、キット；
    （１）WFA／VVAレクチン、
    （２）CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体。     A lectin-antibody sandwich assay kit for detecting or quantifying CSF1R containing a WFA / VVA-binding sugar chain, comprising (1) and (2);
    (1) WFA / VVA lectin,
    (2) CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13 At least one anti-CSF1R antibody selected from the group consisting of antibodies, CSR-26, CSR-27, and CSR-29.
  18. 　さらに、以下の（３）を含む、請求項１７に記載のキット；
    （３）WFA／VVA結合性糖鎖含有CSF1R及び／又はWFA／VVA結合性糖鎖非含有CSF1Rからなる標準物質。     Furthermore, the kit of Claim 17 containing the following (3);
    (3) A standard substance comprising a CSF1R containing a WFA / VVA-binding sugar chain and / or a CSF1R containing no WFA / VVA-binding sugar chain.
  19. 　WFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンである、請求項１８又は１９に記載のキット。 The kit according to claim 18 or 19, wherein the WFA / VVA lectin is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA.
  20. 　レクチン－抗体サンドイッチアッセイが、被験者由来の体液試料に適用し、体液試料中のWFA／VVA結合性糖鎖含有CSF1Rを検出又は定量するためのアッセイである、請求項１７～１９のいずれか一項に記載のキット。 The lectin-antibody sandwich assay is an assay for applying to a body fluid sample derived from a subject and detecting or quantifying CSF1R containing a WFA / VVA-binding sugar chain in the body fluid sample. The kit according to 1.
  21. 　CSR-3（国際受託番号：NITE BP-02117）、CSR-4（国際受託番号：NITE BP-02118）CSR-18（国際受託番号：NITE BP-02119）、CSR-21（国際受託番号：NITE BP-02120）、CSR-30（国際受託番号：NITE BP-02121）からなる群から選択されるいずれか１つのハイブリドーマから産生される抗CSF1R抗体又はその抗体結合性フラグメント。 CSR-3 (International accession number: NITE BP-02117), CSR-4 (International accession number: NITE BP-02118) CSR-18 (International accession number: NITE BP-02119), CSR-21 (International accession number: NITE) BP-02120), CSR-30 (international accession number: NITE BP-02121), an anti-CSF1R antibody or an antibody-binding fragment thereof produced from any one hybridoma selected from the group consisting of:
  22. 　肝硬変患者である被験者の肝細胞がん発症リスク値及び／又は予後判定値を算出するためのキットであって、（１）及び（２）のレクチンを含むことを特徴とするキット；
    （１）WFA／VVAレクチン、
    （２）CSF1R特異的レクチン。     A kit for calculating a risk value and / or prognosis value for developing hepatocellular carcinoma of a subject who is a cirrhotic patient, the kit comprising the lectin according to (1) and (2);
    (1) WFA / VVA lectin,
    (2) CSF1R specific lectin.
  23. 　さらに、（３）を含むことを特徴とする請求項２２に記載のキット；
    （３）抗CSF1R抗体又はその抗体結合性フラグメント。     The kit according to claim 22, further comprising (3);
    (3) An anti-CSF1R antibody or an antibody-binding fragment thereof.
  24. 　（１）のWFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンであり、
    （２）のCSF1R特異的レクチンが、RCA120, DSA, PHA-E4、SNA、SSA、TJA-I、LEL、STL及びConAからなる群から選択される少なくとも１つのレクチンである，請求項２２又は２３に記載のキット。     The WFA / VVA lectin of (1) is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA,
    24. The CSF1R-specific lectin of (2) is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA. The kit according to 1.
  25. 　（１）又は（２）のレクチンのいずれか一方がWFA／VVA結合性糖鎖含有CSF1Rの捕捉用に設けられた固相に結合されており、かつ他方が検出用の液相に溶解又は分散されている、請求項２２～２４のいずれか一項に記載のキット。 Either one of the lectins of (1) or (2) is bound to a solid phase provided for capturing WFA / VVA-binding sugar chain-containing CSF1R, and the other is dissolved or dispersed in a liquid phase for detection The kit according to any one of claims 22 to 24, wherein:
  26. 　（１）及び（２）のレクチンの両方が同一もしくは異なるレクチンアレイ上に結合している、請求項２２～２４のいずれか一項に記載のキット。 The kit according to any one of claims 22 to 24, wherein both (1) and (2) lectins are bound on the same or different lectin arrays.
  27. 　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するためのキットであって、（１）～（３）を含むことを特徴とする判定用キット；
    （１）WFA／VVAレクチン、
    （２）CSF1R特異的レクチン。
    （３）抗CSF1R抗体又はその抗体結合性フラグメント。     A kit for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who has cirrhosis, comprising (1) to (3);
    (1) WFA / VVA lectin,
    (2) CSF1R specific lectin.
    (3) An anti-CSF1R antibody or an antibody-binding fragment thereof.
  28. 　（１）のWFA／VVAレクチンが、天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンであり、
    （２）のCSF1R特異的レクチンが、RCA120, DSA, PHA-E4、SNA、SSA、TJA-I、LEL、STL及びConAからなる群から選択される少なくとも１つのレクチンであり，及び
    （３）の抗CSF1R抗体が、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体である、請求項２７に記載の判定用キット。     The WFA / VVA lectin of (1) is at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA,
    (2) the CSF1R-specific lectin is at least one lectin selected from the group consisting of RCA120, DSA, PHA-E4, SNA, SSA, TJA-I, LEL, STL, and ConA, and (3) Anti-CSF1R antibodies are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR The determination kit according to claim 27, wherein the determination kit is at least one anti-CSF1R antibody selected from the group consisting of -13, CSR-26, CSR-27, and CSR-29 antibodies.
  29. 　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するための方法であって、（１）及び（２）の工程を含むことを特徴とする方法；
    （１）被験者由来の体液試料から抗CSF1R抗体を用いてCSF1Rタンパク質を分離精製する工程、
    （２）（１）で分離精製されたCSF1R上のWFA／VVA結合性糖鎖含有量及びCSF1R特異的糖鎖含有量を、測定する工程。     A method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a cirrhotic patient, comprising the steps of (1) and (2);
    (1) a step of separating and purifying CSF1R protein from a body fluid sample derived from a subject using an anti-CSF1R antibody,
    (2) A step of measuring the WFA / VVA-binding sugar chain content and the CSF1R-specific sugar chain content on the CSF1R separated and purified in (1).
  30. 　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するための方法であって、（１）及び（２）の工程を含むことを特徴とする方法；
    （１）被験者由来の体液試料から天然WFA、リコンビナントWFA、単量体リコンビナントWFA、及びVVAからなる群から選択される少なくとも１つのレクチンを用いてWFA／VVA結合性糖鎖を含有する糖タンパク質を分離する工程、
    （２）（１）で分離精製されたWFA／VVA結合性糖鎖を含有する糖タンパク質から抗CSF1R抗体を用いてCSF1Rタンパク質を検出又は定量する工程、
    ここで、抗CSF1R抗体は、CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体である。     A method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who is a cirrhotic patient, comprising the steps of (1) and (2);
    (1) A glycoprotein containing a WFA / VVA-binding sugar chain using at least one lectin selected from the group consisting of natural WFA, recombinant WFA, monomeric recombinant WFA, and VVA from a body fluid sample derived from a subject. Separating,
    (2) a step of detecting or quantifying the CSF1R protein using the anti-CSF1R antibody from the glycoprotein containing the WFA / VVA-binding sugar chain separated and purified in (1),
    Here, anti-CSF1R antibodies are CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR- 9, at least one anti-CSF1R antibody selected from the group consisting of CSR-13, CSR-26, CSR-27, and CSR-29 antibodies.
  31. 　肝硬変患者である被験者の肝細胞がん発症リスク及び／又は予後を判定するための方法であって、被験者由来の体液試料に対し、（１）及び（２）を用いたレクチン－抗体サンドイッチアッセイを行う工程を含むことを特徴とする方法；
    （１）WFA／VVAレクチン、
    （２）CSR-3、CSR-4、CSR-18、CSR-21、CSR-30、CSR-5、CSR-6、CSR-22、CSR-24、CSR-7、CSR-9、CSR-13、CSR-26、CSR-27、及びCSR-29抗体からなる群から選択される少なくとも１つの抗CSF1R抗体。     A method for determining the risk and / or prognosis of hepatocellular carcinoma in a subject who has cirrhosis, wherein a lectin-antibody sandwich assay using (1) and (2) is performed on a body fluid sample derived from the subject. A method comprising the step of performing;
    (1) WFA / VVA lectin,
    (2) CSR-3, CSR-4, CSR-18, CSR-21, CSR-30, CSR-5, CSR-6, CSR-22, CSR-24, CSR-7, CSR-9, CSR-13 At least one anti-CSF1R antibody selected from the group consisting of antibodies, CSR-26, CSR-27, and CSR-29.
  32. 　肝細胞がん未罹患肝硬変患者における肝細胞がん発症リスクを判定するための方法であって、（１）及び（２）の工程を含むことを特徴とする方法；
    （１）肝細胞がん未罹患肝硬変患者である被験者から採取された体液試料中のWFA／VVA結合性糖鎖を含有するCSF1R量、及び全CSF1R量を測定する工程、
    （２）（１）で得られた測定値をもとに、全CSF1Rに占めるWFA／VVA結合性糖鎖を有するCSF1R量の比率を算出する工程、
    （３）あらかじめ十分な母数の肝細胞がん未罹患肝硬変患者から採取された体液試料に対し、前記（１）及び（２）と同じ工程により、全CSF1Rに占めるWFA／VVA結合性糖鎖を有するCSF1R量の比率を全患者についてそれぞれ算出する工程、
    （４）（３）で用いた全患者をフォローアップして得た発がん率データに基づいて最適カットオフ値を算出する工程、
    （５）（２）で算出された被験者の比率の値を（４）で算出された最適カットオフ値と比較して上回っている場合に肝細胞がん発症リスクが高いと判定する方法。     A method for determining the risk of developing hepatocellular carcinoma in a cirrhosis patient who does not have hepatocellular carcinoma, comprising the steps of (1) and (2);
    (1) a step of measuring the amount of CSF1R containing a WFA / VVA-binding sugar chain and the total amount of CSF1R in a body fluid sample collected from a subject who is a liver cirrhosis patient who does not have hepatocellular carcinoma;
    (2) calculating the ratio of the amount of CSF1R having a WFA / VVA-binding sugar chain to the total CSF1R based on the measurement value obtained in (1),
    (3) A WFA / VVA-binding glycan occupying in the total CSF1R is obtained from a bodily fluid sample collected from a cirrhosis patient who does not have hepatocellular carcinoma in advance by the same process as (1) and (2) above. Calculating the ratio of CSF1R amount for all patients,
    (4) calculating an optimal cutoff value based on carcinogenicity data obtained by following up all patients used in (3);
    (5) A method for determining that the risk of developing hepatocellular carcinoma is high when the value of the ratio of subjects calculated in (2) is higher than the optimum cutoff value calculated in (4).
PCT/JP2016/077683 2015-09-18 2016-09-20 Method for predicting prognosis and risk of developing hepatocellular carcinoma in liver cirrhosis patient WO2017047813A1 (en)

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