WO2017047813A1 - 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 - Google Patents

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 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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English (en)
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/zh
Priority to JP2017540047A priority patent/JP6779504B2/ja
Publication of WO2017047813A1 publication Critical patent/WO2017047813A1/ja
Priority to HK18114481.7A priority patent/HK1255344A1/zh

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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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PCT/JP2016/077683 2015-09-18 2016-09-20 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 WO2017047813A1 (ja)

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