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WO2023068284A1 - Procédé et kit de diagnostic ou de détection du cancer du rein - Google Patents

Procédé et kit de diagnostic ou de détection du cancer du rein Download PDF

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Publication number
WO2023068284A1
WO2023068284A1 PCT/JP2022/038841 JP2022038841W WO2023068284A1 WO 2023068284 A1 WO2023068284 A1 WO 2023068284A1 JP 2022038841 W JP2022038841 W JP 2022038841W WO 2023068284 A1 WO2023068284 A1 WO 2023068284A1
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Prior art keywords
icam
lectin
cancerous
lectins
cancer
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Japanese (ja)
Inventor
敦 久野
裕之 梶
弥栄 金井
恵吏 新井
賀子 北爪
厚志 松田
大輔 高倉
祥子 大橋
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National Institute of Advanced Industrial Science and Technology AIST
Keio University
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National Institute of Advanced Industrial Science and Technology AIST
Keio University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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 methods and kits for diagnosing or detecting renal cancer, and particularly to methods and kits for diagnosing or detecting renal cancer using lectins.
  • ICAM-1 intercellular adhesion molecule 1 expressed on the surface of vascular endothelial cells and immune system cells is known (see, for example, Non-Patent Document 1, etc.). reference).
  • ICAM-1 is a transmembrane protein with one transmembrane domain, an N-terminal extracellular domain and a C-terminal cytoplasmic domain. ICAM-1 serves as a binding site for various ligand proteins.
  • ICAM-1 binds to immune-related ligands, particularly proteins commonly expressed in endothelial cells and leukocytes such as LFA-1, and increases extravasation. It is known that it promotes the process of leukocyte migration across the vascular endothelium, such as embolization and inflammatory response.
  • Non-Patent Document 2 focuses on the sugar chain structure of ICAM-1 in endothelial cells, and when the sugar chain is a high-mannose sugar chain, the cell adhesion of CD16+ monocytes is enhanced more than in other cases. It is shown that In addition, Non-Patent Document 3 analyzes the sugar chain structure of ICAM-1 in the lesion site of atherosclerosis. -1 is present in lesions of atherosclerosis.
  • sugar chain-binding proteins which are known to specifically bind to each sugar chain structure, are used to detect various sugar chain structures.
  • lectins have been known so far, and each lectin has a different sugar chain structure that exhibits specificity.
  • the present invention has been made in view of the above problems, and its purpose is to make it possible to diagnose or detect specific diseases using detection of the sugar chain structure of ICAM-1.
  • the present inventors have made intensive studies and found that the sugar chain structure of ICAM-1 expressed in cells obtained from renal cancer tissue is similar to that of ICAM-1 expressed in normal cells.
  • the present invention was completed by discovering that it is different from the sugar chain structure.
  • the method for diagnosing renal cancer according to the present invention includes CGL2, WFA, ECA, WGA, CLA, STL, BPL, Discoidin II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN , CBA-pro (CBA precursor), UEA-I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and at least one lectin selected from the group consisting of MPA1 with a biological sample collected from a subject quantifying ICAM-1 bound to said lectin in said biological sample; and diagnosing kidney cancer in said subject according to the amount of ICAM-1 bound to said lectin.
  • a method comprising:
  • the method for detecting renal cancer according to the present invention includes CGL2, WFA, ECA, WGA, CLA, STL, BPL, Discoidin II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN, CBA - contacting at least one lectin selected from the group consisting of pro, UEA-I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1 with a biological sample taken from a subject; quantifying ICAM-1 bound to said lectin in a biological sample; and detecting renal cancer in said subject based on comparing the amount of ICAM-1 bound to said lectin with a predetermined reference value. and determining whether the group consisting of pro, UEA-I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1 with a biological sample taken from a subject; quantifying ICAM-1 bound to said lectin in a biological sample; and detecting renal cancer in said
  • Each of the above methods utilizes the fact that the sugar chain structures of ICAM-I in renal cancer tissue and non-renal cancer tissue found by the present inventors are different. Specifically, in the above lectin, the specificity of sugar chains of ICAM-1 in renal cancer tissue and ICAM-1 in non-renal cancer tissue is significantly different, and this difference is utilized to detect renal cancer. diagnosing or detecting. In each of the above methods, renal cancer is diagnosed or detected based on the remarkable difference in the reactivity of the lectin to ICAM-1 between renal cancer tissue and non-renal cancer tissue, so that the diagnosis can be performed accurately and easily. Or detection can be performed.
  • kidney tissue, blood or urine can be used as the biological sample.
  • the step of quantifying ICAM-1 can be performed using a probe that specifically binds to ICAM-1.
  • Each of the above methods may further comprise a step of concentrating the biological sample before contacting the lectin with the biological sample.
  • the diagnostic kit for renal cancer includes CGL2, WFA, ECA, WGA, CLA, STL, BPL, Discoidin II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN, CBA-pro , UEA-I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1, and a probe that specifically binds to ICAM-1.
  • the above lectin having remarkably different specificities for sugar chains of ICAM-1 in renal cancer tissue and ICAM-1 in non-renal cancer tissue, and ICAM- lectin-bound ICAM-1 in biological samples and ICAM-1 in non-cancer tissues, respectively. Therefore, each of the above methods can be easily performed by using the kit.
  • the probe preferably contains an antibody, peptide, or aptamer that specifically binds to ICAM-1.
  • the method and kit for diagnosing or detecting renal cancer According to the method and kit for diagnosing or detecting renal cancer according to the present invention, it is possible to accurately and simply diagnose or detect renal cancer in a subject using a biological sample of the subject.
  • FIG. 1 shows the results of Western blotting of ICAM-1-enriched tissues obtained by immunoprecipitation using an anti-ICAM-1 antibody in Example 1.
  • 2 is a diagram showing a list of lectins mounted on the lectin array chip (LecChip ver1.0) used in Example 1.
  • FIG. 3 is a diagram showing a list of lectins mounted on the lectin array chip (LecChip ver 2.0) used in Example 1.
  • FIG. 4 is a diagram showing the results of lectin array analysis for ICAM-1-enriched tissue obtained by immunoprecipitation using anti-ICAM-1 antibody performed in Example 1.
  • FIG. 5 shows the results of lectin array analysis for ICAM-1-enriched tissue obtained by immunoprecipitation using anti-ICAM-1 antibody in Example 1.
  • FIG. 6 shows the results of lectin array analysis for tissue enriched with ICAM-1 obtained by immunoprecipitation using an anti-ICAM-1 antibody in Example 1.
  • FIG. 7 is a graph of lectins showing a significant difference in statistical analysis after the lectin array analysis shown in FIGS. 4-6 performed in Example 1.
  • FIG. Specifically, from the results shown in FIGS. 4 to 6, some of the lectins that are significantly elevated in the cancerous area (Tumor) compared to the non-cancer area (Normal) were extracted, and each lectin recognizing a high mannose type sugar chain was selected. Results for groups (rKAA, rMPA1, rMPA2, rESA2) are shown. In each graph, the vertical axis indicates the relative signal value (Relative Intensity (%)). 8 is a graph of lectins showing a significant difference in statistical analysis after the lectin array analysis shown in FIGS.
  • FIG. 4-6 performed in Example 1.
  • FIG. 9 is a diagram showing the results of Western blotting of ICAM-1-enriched tissues collected using a cancer-part-specific lectin performed in Example 1.
  • FIG. 10 shows the results of comparative glycan analysis of tissue enriched with ICAM-1 by mass spectrometry performed in Example 1.
  • FIG. 11 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver1.0 performed in Example 2.
  • FIG. 12 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver1.0 performed in Example 2.
  • FIG. 12 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver1.0 performed in Example 2.
  • FIG. 13 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver1.0 performed in Example 2.
  • FIG. 14 shows the results of multi-specimen comparative sugar chain analysis of ICAM-1-enriched tissues in the lectin array chip ver1.0 performed in Example 2.
  • FIG. 14 shows the results of multi-specimen comparative sugar chain analysis of ICAM-1-enriched tissues in the lectin array chip ver1.0 performed in Example 2.
  • ICAM-1 enriched lectin array analysis results obtained from non-cancerous and cancerous tissue samples of 99 renal cancer patients recruited for analysis were summarized for each lectin. It is a boxplot. The distribution of 8 out of 31 lectins showing significant binding signals is shown by dividing them into non-cancer areas (left) and cancer areas (right), and the vertical axis indicates the relative signal value.
  • FIG. 15 shows the results of multi-specimen comparative sugar chain analysis of ICAM-1-enriched tissue in the lectin array chip ver2.0 performed in Example 2.
  • FIG. Specifically, ICAM-1 enriched lectin array analysis results obtained from non-cancerous and cancerous tissue samples of 99 renal cancer patients recruited for analysis were summarized for each lectin. It is a boxplot.
  • FIG. 16 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver 2.0 performed in Example 2.
  • FIG. 16 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver 2.0 performed in Example 2.
  • FIG. 17 shows the results of multi-specimen comparative sugar chain analysis of tissue enriched with ICAM-1 using the lectin array chip ver 2.0 performed in Example 2.
  • FIG. 18 shows the results of multi-specimen comparative sugar chain analysis of ICAM-1-enriched tissue in the lectin array chip ver2.0 performed in Example 2.
  • ICAM-1 enriched lectin array analysis results obtained from non-cancerous and cancerous tissue samples of 99 renal cancer patients recruited for analysis were summarized for each lectin. It is a boxplot. 7 out of 32 lectins showing significant binding signals are divided into non-cancerous areas (left) and cancerous areas (right), and their distributions are shown. The vertical axis indicates relative signal values.
  • FIG. 19 shows the results of hierarchical clustering of cancerous areas (white) and non-cancerous areas (black) in 99 cases of renal cancer patients using the fluorescence intensities of 90 types of lectins performed in Example 2.
  • FIG. 4 is a diagram showing; 20 is a diagram showing the results of receiver operating characteristic (ROC) analysis using data of 99 cases of renal cancer patients performed in Example 2.
  • ROC receiver operating characteristic
  • FIG. 1 receiver operating characteristic
  • FIG. 22 shows the results of multi-specimen comparative sugar chain analysis of tissue lysate (crude glycoprotein solution) by lectin array using lectin array chip ver1.0 performed in Example 2.
  • FIG. 23 is a diagram showing the results of multi-specimen comparative sugar chain analysis of tissue lysate (crude glycoprotein solution) by lectin array using lectin array chip ver1.0 performed in Example 2.
  • a whisker diagram is shown. In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right).
  • 24 is a diagram showing the results of multi-specimen comparative sugar chain analysis of tissue lysate (crude glycoprotein solution) by lectin array using lectin array chip ver1.0 performed in Example 2.
  • FIG. Specifically, a box summarizing the lectin array analysis results of crude glycoprotein solutions obtained from non-cancerous and cancerous tissue samples in frozen tissue specimens of 99 cases of renal cancer patients recruited for analysis by lectin.
  • a whisker diagram is shown. In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right).
  • FIG. 25 is a diagram showing the results of multi-specimen comparative sugar chain analysis of tissue lysate (crude glycoprotein solution) by lectin array using lectin array chip ver1.0 performed in Example 2.
  • FIG. Specifically, a box summarizing the lectin array analysis results of crude glycoprotein solutions obtained from non-cancerous and cancerous tissue samples in frozen tissue specimens of 99 cases of renal cancer patients recruited for analysis by lectin. A whisker diagram is shown. In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right).
  • FIG. 26 shows the lectin array analysis results (Crude: left) of crude glycoprotein solutions of frozen tissues of 99 cases of renal cancer patients performed in Example 2 and the lectin array analysis results for ICAM-1-enriched crude glycoprotein solutions. (Right), and is a boxplot showing the results of the lectin group in which a particularly remarkable divergence occurred (significantly high value in the cancerous part). In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right).
  • FIG. 27 shows the lectin array analysis results of crude glycoprotein solutions of frozen tissues of 99 cases of renal cancer patients (Crude: left) and lectin array analysis results for ICAM-1 enriched crude glycoprotein solutions in Example 2.
  • FIG. 28 shows the lectin array analysis results (Crude: left) of crude glycoprotein solutions of frozen tissues of 99 cases of renal cancer patients performed in Example 2 and the lectin array analysis results for ICAM-1 enrichment in crude glycoprotein solutions.
  • (Right) is a diagram comparing the results of the lectin group that produces mutually correlated signals but shows high values in cancerous areas. In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right).
  • FIG. 29 shows the lectin array analysis results (Crude: left) of crude glycoprotein solutions of frozen tissues of 99 cases of renal cancer patients performed in Example 2 and the lectin array analysis results for ICAM-1 enrichment in crude glycoprotein solutions.
  • (Right) is a diagram comparing the results of the lectin group that produces mutually correlated signals but has high values in non-cancerous areas. In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right).
  • FIG. 30 shows the results of estimating sugar chain units recognizing galactose/N-acetylgalactosamine recognizing lectins, based on lectin array analysis of enzymatic digests of ICAM-1 enriched isolated from patient frozen tissues performed in Example 2.
  • Example 31 shows the results of estimating sugar chain units recognizing galactose/N-acetylgalactosamine recognizing lectins by lectin array analysis of enzymatic digests of ICAM-1 enriched isolated from patient frozen tissues performed in Example 2. It is a figure which shows.
  • One case in which the WFA signal was negative was randomly selected from the frozen tissue samples of renal cancer patients, and ICAM-1 was immunoprecipitated (IP), treated with an enzyme, and added to a lectin array.
  • FIG. 32 shows a two-dimensional plot prepared using CLA signal data (horizontal axis) against signal data (vertical axis) for each of the two lectins, WFA and RCA120, obtained in Example 3.
  • FIG. Each signal data is the signal data of the lectin array (LecChip ver1.0 and 2.0) obtained in Example 2.
  • White plots are signal data of non-cancerous sites, and black plots are signal data of cancer sites.
  • FIG. 33 shows a two-dimensional plot prepared using CLA signal data (horizontal axis) against signal data (vertical axis) for each of the two lectins, ECA and Discoidin II, obtained in Example 3.
  • FIG. Each signal data is the signal data of the lectin array (LecChip ver1.0 and 2.0) obtained in Example 2.
  • White plots are signal data of non-cancerous sites, and black plots are signal data of cancer sites.
  • One embodiment of the present invention is CGL2, WFA, ECA, WGA, CLA, STL, BPL, Discoidin II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN, CBA-pro, UEA-I , KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1, with a biological sample collected from a subject;
  • a method for diagnosing renal cancer comprising the steps of quantifying bound ICAM-1 and diagnosing renal cancer in a subject according to the amount of ICAM-1 bound to lectin.
  • the CLA is preferably a recombinant CLA.
  • the method according to the present embodiment utilizes the fact that renal cancer tissue and non-renal cancer tissue have different sugar chain structures of ICAM-I. Specifically, in the above lectin, the specificity of sugar chains of ICAM-1 in renal cancer tissue and ICAM-1 in non-renal cancer tissue is significantly different, and this difference is utilized to detect renal cancer. Diagnose.
  • the characteristics such as the origin and sugar chain specificity of the lectin used in the present embodiment are described in, for example, International Publication No. 2012/133127, "Lectin Frontier DataBase (LfDB) (http://acgg.asia/lfdb2/)", etc. It is described in. In addition to these, the origin of ESA2 in particular is described in Glycobiology vol. 17 no. 5 pp. 479-491, 2007, and for CFA sub1, Asian Pacific International Marine Biotechnology Conference (May 21, 2017. Hawaii, URL: https://cms.ctahr.hawaii.finaledu/Portals/235/apmbc.abstracts pdf) has been published.
  • the above lectins are also available from, for example, EY Laboratories, Vector Laboratories, Merck, Fujifilm Wako Pure Chemical Industries, Ltd., and all of them are well-known lectins.
  • the lectins used in this embodiment may be of natural origin and from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells and mammalian cells. It may be a recombinant lectin as a product produced by recombinant technology.
  • the amino acid sequence of the lectin used in this embodiment does not have to be full-length as long as it retains the recognition site for the target sugar chain in ICAM-1. good too.
  • within the range that does not impair the ability to recognize a specific target sugar chain for example, less than 10%, preferably less than 5%, more preferably less than 3%, particularly preferably less than 1% amino acid residue in the amino acid sequence deletions, substitutions, additions or insertions of Deletion, substitution, addition or insertion at that time is preferably only at the C-terminus or N-terminus of the amino acid sequence of the lectin.
  • the subject is a human or animal, including mice, guinea pigs, rats, monkeys, dogs, cats, hamsters, horses, cows and pigs, preferably humans.
  • the biological sample is solid tissue and body fluid derived from the subject, preferably renal tissue, blood or urine.
  • This embodiment may further include a step of concentrating the biological sample before contacting the lectin with the biological sample.
  • concentration method is not particularly limited as long as it is a method commonly used in this technical field, and for example, an immunoprecipitation method using an anti-ICAM-1 antibody can be used.
  • the step of bringing the lectin into contact with the biological sample collected from the subject can be performed, for example, by adding and mixing a solution containing the lectin to the sample solution.
  • the biological sample is renal tissue
  • the renal tissue can be solubilized and the solution containing the lectin is added and mixed as shown in the examples below.
  • the lectin used here may contain biotin, a His tag, or the like, so that only the ICAM-1 bound to the lectin can be recovered in the subsequent step of quantifying the ICAM-1 bound to the lectin.
  • a labeling molecule commonly used in this technical field may be attached.
  • the lectin in the step of quantifying ICAM-1 bound to the lectin in the biological sample, first, the lectin is brought into contact with the biological sample, and then the lectin is recovered. For this reason, the biotinylated lectin can be recovered using streptavidin-immobilized magnetic beads, for example, by using a biotinylated lectin as in Examples shown later, although not limited to the following technique. Subsequently, ICAM-1 bound to the lectin can be quantified by using a molecule capable of specifically binding to ICAM-1, such as the anti-ICAM-1 antibody described above.
  • molecules that can specifically bind to ICAM-1 are not limited to antibodies, and peptides, aptamers, and the like may also be used.
  • each step can be carried out on the chip using a lectin array chip in which lectins are pre-arranged on the chip as used in the examples below. This is convenient and preferable.
  • a lectin bead array such as the method of Shimazaki et al. (Shimazaki H et al. Analytical Chemistry 91 (17), 11162-11169 (2019)) using lectin-bound beads can be used.
  • the step of diagnosing the subject's renal cancer is performed based on the above quantitative results. If the diagnosis is made based on the quantification results, it is not limited to the following method.
  • a subject can be diagnosed as having kidney cancer if it is significantly different compared to the quantified value in .
  • the method is not particularly limited as long as the comparison can clearly distinguish the difference from the control, but the absolute value may be used as the measured value of each sample as in Example 3 below, or as in Example 2 below.
  • a relative value normalized by the average value of multiple lectin results may be used.
  • a reference value is established based on the measurement value in a non-cancerous part that is a control, and the measurement value in the biological sample of the subject exceeds the reference value.
  • a subject may be diagnosed as having kidney cancer in some cases.
  • the measured value of the biological sample of the subject is, for example, 10% or more, preferably 20% or more, more preferably 30% or more, and particularly preferably 50% or more compared to the measured value of the control such as non-cancerous tissue. % or higher, the subject may be diagnosed as having kidney cancer.
  • the relative values obtained for each of the plurality of lectins for the non-cancerous region serving as a control are compared with each other, and if lectins with significantly different relative values are included, the subject may be diagnosed as having renal cancer.
  • inventions include CGL2, WFA, ECA, WGA, CLA, STL, BPL, Discoidin II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN, CBA-pro, UEA- contacting at least one lectin selected from the group consisting of I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1 with a biological sample collected from a subject; and the lectin in the biological sample. and determining whether or not renal cancer is detected in the subject based on comparison of the amount of ICAM-1 bound to the lectin with a predetermined reference value. , a method for the detection of renal cancer.
  • the CLA is preferably a recombinant CLA.
  • the lectin and biological sample used in this embodiment can be the same as those shown in the embodiment of the method for diagnosing renal cancer, and the lectin and the biological sample are brought into contact with each other.
  • the steps and the step of quantifying ICAM-1 bound to the lectin can also use the technique shown in the embodiment of the method for diagnosing renal cancer.
  • the step of determining whether or not renal cancer is detected is performed based on the comparison of the amount of ICAM-1 bound to lectin with a predetermined reference value, as described above.
  • the amount of ICAM-1 bound to the lectin in the target biological sample is determined using the quantified value in the control such as non-cancer tissue as the reference value. If the quantified value is significantly different from the reference value, it can be determined that kidney cancer has been detected in the subject.
  • the method is not particularly limited as long as the comparison can clearly distinguish the difference from the control, but the absolute value may be used as the measured value of each sample as in Example 3 below, or as in Example 2 below.
  • a relative value normalized by the average value of multiple lectin results may be used.
  • a reference value is established based on the measurement value in a non-cancerous part that is a control, and the measurement value in the biological sample of the subject exceeds the reference value. It may be determined that kidney cancer is detected in the subject in some cases.
  • the measured value of the biological sample of the subject is, for example, 10% or more, preferably 20% or more, more preferably 30% or more, and particularly preferably 50% or more compared to the measured value of the control such as non-cancerous tissue. % or higher, it may be determined that renal cancer has been detected in the subject.
  • the relative values obtained for each of the plurality of lectins for the non-cancerous region serving as a control for example, the relative values obtained for each of the plurality of lectins for the non-cancerous region serving as a control, and the plurality for the biological sample of the subject and the relative values obtained for each of the lectins are compared with each other, and when lectins with significantly different relative values are included, it may be determined that renal cancer has been detected in the subject.
  • the CLA is preferably a recombinant CLA.
  • the above lectin having remarkably different sensitivity and specificity to sugar chains of ICAM-1 in renal cancer tissue and ICAM-1 in non-renal cancer tissue specifically binds to ICAM-1. Since it contains a probe that does, it is possible to quantify ICAM-1 bound to lectin in a biological sample and ICAM-1 in non-cancer tissue, respectively. It can be done easily.
  • a probe that specifically binds to ICAM-1 can be, for example, an antibody, peptide or aptamer that specifically binds to ICAM-1. As mentioned above, in the case of an antibody, it is preferably a monoclonal antibody.
  • Example 1 Exploratory experiment on changes in sugar chains on ICAM-1
  • the total protein amount in the tissue lysate was quantified using Micro BCA Protein Assay Kit (manufactured by ThermoFisher) based on the conventional BCA method.
  • immunoprecipitation was performed according to the method of Kuno et al. and Wagatsuma et al. (Kuno A et al. Molecular & Cellular Proteomics 8 (1), 99-108 (2009). , Wagatsuma T et al Frontiers in Oncology 10, 338 (2020)).
  • ICAM. -1 eluate (ICAM-1 enriched).
  • a half volume of the eluate was used for western blotting according to a conventional method.
  • a 10-20% gradient polyacrylamide gel was used for the electrophoresis gel, the biotinylated antibody described above was used as the primary detection antibody, and streptavidin-HRP and a substrate for detection (ImmnoStarLD, manufactured by Fujifilm Wako) were used for detection. was used. The results are shown in FIG.
  • Each sample (ICAM-1-enriched) was applied to the lectin array chips ver1.0 and ver2.0 with the immunoprecipitated solution equivalent to 10 ⁇ g of total protein, and subjected to binding reaction at 20° C. for 10 hours or longer. .
  • 2 ⁇ L of a human serum-derived IgG solution (manufactured by Sigma-Aldrich) was added and allowed to react for 30 minutes. Then, 100 ng of the above biotinylated antibody was added and reacted at 20°C for 1 hour, then 200 ng of Cy3-labeled streptavidin was added and reacted at 20°C for 30 minutes.
  • ICAM-1 is present in cancerous and non-cancerous tissues of renal cancer patients, but there are differences in the sugar chain structure, and these differences are caused by specific lectins on the lectin array. It turned out to be explainable.
  • a group of lectins that preferentially bind to ICAM-1 present in cancerous areas will be referred to as cancerous specific lectins.
  • lectin precipitation 10 ⁇ L of the magnetic beads and 2 ⁇ g of biotin-labeled lectin were reacted to prepare lectin-immobilized magnetic beads and used.
  • tissue lysate equivalent to 20 ⁇ g of total protein was added, binding reaction was carried out, unbound substances were washed with PBSTx, and 10 ⁇ L of TBS buffer containing 0.2% SDS was added to bind glycoprotein molecules. Elution was performed by heat treatment at 95° C. for 10 minutes. This is used as the rKAA-binding protein solution.
  • molecules that bind to KAA are also present in the rKAA-binding protein solution.
  • ICAM-1 was enriched by the immunoprecipitation method described above and the presence of ICAM-1 in the eluate was confirmed by Western blot. The results are shown in FIG.
  • FIG. 10 shows the analysis results of N-type glycosylation to asparagine at position 406 of ICAM-1.
  • the latter is also known to bind to rKAA, rMPA1, rMPA2, rESA2, etc. in FIG. From the above, we were able to verify the response validity of the cancer-specific lectins obtained by the lectin array by comparative glycan analysis using mass spectrometry.
  • Example 2 Validation test of ICAM-1 epicancer-specific sugar chain changes
  • a value standardized by the average value of signals obtained from each of the 45 lectins on each lectin array chip is used.
  • the results for lectin array chip ver1.0 are shown in FIGS. 15-18, respectively.
  • Hierarchical clustering analysis of acquired data In order to investigate how the results of the 198 samples are classified according to the characteristic sugar chain profiles obtained from the above analysis, using the fluorescence intensity data of the 90 lectins used in the above lectin array, Hierarchical clustering was performed on 99 cancerous tissue samples and 99 non-cancer tissue samples from 99 renal cancer patients from whom appropriate data could be obtained. The results are shown in FIG.
  • FIGS. 20 and 21 show that cancerous and non-cancerous areas can be clearly separated by examining the sugar chains on ICAM-1.
  • ROC analysis was performed using cancerous and non-cancerous areas of 99 cases of renal cancer patients. The results are shown in FIGS. 20 and 21.
  • FIG. 20 and 21 only lectins with AUC>0.8 are shown, and lectins with signals below the lower limit of the quantification range are shown in italics. These correspond to the lectins omitted from FIGS.
  • lectins other than the lectins shown in italics which are significantly higher in cancerous areas (T) than in non-cancerous areas (N), are shown in bold.
  • FIGS. 20 and 21 there are 14 lectins with an AUC of 0.95 or higher that distinguish between cancerous and non-cancerous lesions, of which 7 (rWFA, ECA, WFA, His-rCLA, BPL , rDiscoidin II, RCA120) was a lectin that gave a significantly stronger signal in cancerous areas.
  • Figures 20 and 21 show 36 lectins with an AUC of 0.8 or higher that can distinguish cancerous from non-cancerous. 27 species (of which 16 species increased in signal at the cancer site (bold)) could be selected.
  • the protein solution was added to the probing buffer so that the total protein amount was 15 ng equivalent to 60 ⁇ L, added to one well of the lectin array chip ver1.0, and reacted at 20° C. for 10 hours or more. .
  • unbound components were washed with PBSTx, and binding signals were detected by array scanning with GlycoStation Reader 1200 (manufactured by Glyco Technica).
  • the signal of each lectin was normalized by the average value of the signals of 45 kinds of lectins according to a conventional method. The results for the 99 cases are shown in Figures 22-25.
  • ICAM-1 significantly decreases the signal in the cancerous part, but the crude glycoprotein solution increases the signal in the cancerous part.
  • Three species UEAI, EEL, and HPA were selected this time, and all recognized the ABO blood group sugar chain antigen.
  • the phenomenon of the appearance of ABO blood group sugar chain antigens on specific proteins in normal tissues and their disappearance due to carcinogenesis has so far been observed in comparative sugar chain analysis of the specific glycoprotein molecule Basigin in pancreatic cancer patient tissues. (Wagatsuma T et al. Frontier in Oncology 10, 338 (2020)).
  • none of the above four lectin signals, which are enhanced in cancer was observed on Basigin in pancreatic cancer tissue.
  • sialidase A which is a sialic acid-degrading enzyme
  • sialidase A which is a sialic acid-degrading enzyme
  • many of these lectins recognize galactose to N-acetylgalactosamine.
  • it recognizes ⁇ 1,3-1,4-linked Gal as the binding position. Therefore, this time, we decided to perform ⁇ -galactosidase digestion to see if a decrease in signal was observed. Since this is digested with galactosidase without digestion with sialidase, the decrease in signal due to this can be proved to have ⁇ 1,4 galactose at the end.
  • the signal of sialic acid-recognizing lectins is relatively weak in the untreated state (black), and the asialosaccharide Chain recognition lectins (ECA, RCA120, BPL, WFA) were relatively high.
  • ECA asialosaccharide Chain recognition lectins
  • the lectin recognizing sialic acid decreased in response, but the lectin recognizing asialo-glycans did not change significantly.
  • ⁇ 1,4-galactosidase digestion was performed while sialic acid was retained, the asialo-glycan-recognizing lectin lost its signal.
  • ICAM-1 in this case has very little terminal sialic acid modification and ⁇ 1,4Gal is exposed.
  • Fig. 31 performed as a control experiment, the signals of the untreated (black) sialic acid-recognizing lectins (MAL-I, SNA, SSA, TJAI) were relatively high and the asialosaccharides other than RCA120 were found to be relatively high. Chain recognition lectins (ECA, BPL, WFA) were relatively low. It is considered that RCA120, unlike others, allows ⁇ 2,6 sialic acid modification of Gal and can bind to it.
  • Example 3 Evaluation of cancer detectability by combination of two lectins
  • the values standardized by the average value of all lectin signals on the lectin array chip were used.
  • the signal data of the lectin array (LecChip ver1.0 and 2.0) obtained in Example 2 were used as they were.
  • kidney cancer can be detected based on the difference from the quantified value of the bound lectin.
  • the lectins used in the present invention have significantly different specificities for the sugar chains of ICAM-1 in renal cancer tissue and ICAM-1 in non-renal cancer tissue. Therefore, according to the method and kit for diagnosing or detecting renal cancer according to the present invention using these lectins, the reactions of the lectins to ICAM-1 in renal cancer tissues and non-renal cancer tissues, respectively It is useful because the diagnosis or detection of renal cancer can be performed accurately and easily by utilizing the gender difference.

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Abstract

Le procédé de diagnostic du cancer du rein selon la présente invention comprend : une étape de mise en contact d'un échantillon biologique prélevé chez un sujet avec au moins une lectine choisie dans le groupe constitué par CGL2, WFA, ECA, WGA, CLA, STL, BPL, discoïdine II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN, CBA-pro, UEA-I, KAA1, ESA2, MPA2, NPA, AAL, jacaline et MPA1 ; une étape de quantification de l'ICAM-1 liée à la lectine dans l'échantillon biologique ; et une étape de diagnostic du cancer du rein chez le sujet sur la base de la quantité d'ICAM-1 liée à la lectine.
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