WO2023068284A1 - Method and kit for diagnosing or detecting kidney cancer - Google Patents

Abstract

The method for diagnosing kidney cancer according to the present invention comprises: a step for contacting a biological sample collected from a subject with at least one lectin selected from the group consisting of 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; a step for quantifying ICAM-1 bound to the lectin in the biological sample; and a step for diagnosing kidney cancer in the subject on the basis of the amount of ICAM-1 bound to the lectin.

Description

Methods and kits for diagnosing or detecting kidney cancer

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.

The sugar chains of complex carbohydrates such as glycoproteins and glycolipids present on cell surfaces and in body fluids function as a kind of information element and are deeply involved in important life phenomena such as development, immunity, cancer, and infection. . As one of such glycoproteins, intercellular adhesion molecule 1 (ICAM-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. For example, 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.

As described above, ICAM-1 is a glycoprotein that is expressed on the cell surface, and various studies have been conducted on the relationship between cells that express ICAM-1 on the surface and the sugar chain structure of the expressed ICAM-1. is being done. For example, 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.

In these studies, sugar chain-binding proteins (lectins), which are known to specifically bind to each sugar chain structure, are used to detect various sugar chain structures. Various lectins have been known so far, and each lectin has a different sugar chain structure that exhibits specificity. Those that bind to chains, complex-type sugar chains or hybrid sugar chains, those that bind to O-glycoside-linked sugar chains (O-type sugar chains) that bind to serine, threonine, etc., and those that bind to fucose-containing sugar chains etc.

Molecular Cell, Vol. 14, 269-276, April 23, 2004. Am J Physiol Heart Circ Physiol 317: H1028-H1038, 2019. PLoS One. 2020 Mar 24; 15(3): e0230358. doi: 10.1371/journal. pone. 0230358. eCollection 2020.

As described above, it has been conventionally known that differences in the sugar chain structure of ICAM-1 expressed on the cell surface are associated with changes in the state and characteristics of cells. Research is being done to make it applicable to diagnosing various diseases. However, the relationship between the sugar chain structure of ICAM-1 and specific cancers has not yet been fully clarified. I haven't been able to.

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.

In order to achieve the above object, 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.

Specifically, 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:

In addition, 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

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.

In each of the above methods, kidney tissue, blood or urine can be used as the biological sample.

In each of the above methods, 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 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 , UEA-I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1, and a probe that specifically binds to ICAM-1.

According to the diagnostic kit for renal cancer according to the present invention, 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.

In the diagnostic kit for renal cancer according to the present invention, the probe preferably contains an antibody, peptide, or aptamer that specifically binds to ICAM-1.

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. FIG. Specifically, the cancer (T) and peripheral non-cancerous (N) tissue lysates of 6 kidney cancer patients ((1) to (6)) were immunoprecipitated using a total protein amount equivalent to 20 μg. , Western blot image obtained by subjecting half of the obtained solution to SDS-polyacrylamide gel electrophoresis. An anti-ICAM-1 antibody is used for detection. Arrows in the figure indicate ICAM-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. 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. Specifically, 2 cases ((1) to (2)) of 6 kidney cancer patients ((1) to (2)) were obtained from cancer (T) and peripheral non-cancerous (N) tissue lysates equivalent to 20 μg as total protein. A half amount of the obtained solution was applied to a lectin array chip, and the image obtained after scanning was graphed. In each graph, the horizontal axis shows 90 kinds of lectins, and the vertical axis shows the relative signal value (Relative Intensity (%)). In the graphs of each case (1) to (2), the upper graph shows the results using the non-cancerous tissue lysate, and the lower graph shows the results using the cancer tissue lysate. 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. Specifically, 2 cases ((3) to (4)) of 6 cases of renal cancer patients ((3) to (4)) of cancerous (T) and surrounding non-cancerous (N) tissue lysates were added to the total protein amount equivalent to 20 μg. A half amount of the obtained solution was applied to a lectin array chip, and the image obtained after scanning was graphed. In each graph, the horizontal axis shows 90 kinds of lectins, and the vertical axis shows the relative signal value (Relative Intensity (%)). In the graphs of each case (3) to (4), the upper graph shows the results using the non-cancerous tissue lysate, and the lower graph shows the results using the cancer tissue lysate. 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. Specifically, 2 cases ((5) to (6)) of 6 cases of renal cancer patients ((5) to (6)) of cancerous (T) and peripheral non-cancerous (N) tissue lysates were added to the total protein amount equivalent to 20 μg. A half amount of the obtained solution was applied to a lectin array chip, and the image obtained after scanning was graphed. In each graph, the horizontal axis shows 90 kinds of lectins, and the vertical axis shows the relative signal value (Relative Intensity (%)). In the graphs of each case (5) to (6), the upper graph shows the results using the non-cancerous tissue lysate, and the lower graph shows the results using the cancer tissue lysate. 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. 4-6 performed in Example 1. FIG. Specifically, from the results shown in FIGS. 4 to 6, some of the lectins significantly elevated in the cancerous area (Tumor) compared to the non-cancer area (Normal) were extracted, and the fucose-recognizing lectin group (AOL, AAL, CLA, ULL) results are shown. In each graph, the vertical axis indicates the relative signal value (Relative Intensity (%)). 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. Specifically, cancer (T) and peripheral non-cancerous (N) tissue lysates of 6 kidney cancer patients ((1) to (6)) were captured with rKAA lectin using a total protein amount equivalent to 20 μg. It is a Western blot image obtained by collecting and further immunoprecipitating with an anti-ICAM-1 antibody, and subjecting half of the obtained solution to SDS-polyacrylamide gel electrophoresis. An anti-ICAM-1 antibody is used for detection. Arrows in the figure indicate ICAM-1. FIG. 10 shows the results of comparative glycan analysis of tissue enriched with ICAM-1 by mass spectrometry performed in Example 1. FIG. Specifically, a comparative glycan analysis was performed using (6) out of the six cases (1) to (6). Figure 2 shows the composition of the N-glycans present. The upper row shows the results for the cancerous part (T), and the lower row shows the results for the non-cancerous part (N). The enhancement of high-mannose-type sugar chains and Lewis-structure-containing complex-type sugar chains encircled in the figure was observed in the cancerous part. 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. 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. 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. 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. 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. Six of the 31 lectins exhibiting significant binding signals are divided into non-cancer areas (left) and cancer areas (right), and their distributions are shown. The vertical axis indicates relative signal values. 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. 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. Nine of the 31 lectins showing significant binding signals are divided into non-cancer (left) and cancerous (right), and their distributions are shown, and the vertical axis indicates the relative signal value. 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. 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. 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. Nine of the 32 lectins showing significant binding signals are divided into non-cancer areas (left) and cancer areas (right), and their distributions are shown, and the vertical axis indicates the relative signal value. 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. 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. The distribution of 8 out of 32 lectins showing significant binding signals is shown separately for non-cancer (left) and cancerous (right), and the vertical axis indicates the relative signal value. 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. 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. The distribution of 8 out of 32 lectins showing significant binding signals is shown separately for non-cancer (left) and cancerous (right), and the vertical axis indicates the relative signal value. 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. 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. 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. FIG. Specifically, ROC analysis was performed using lectin array analysis data of cancerous (T) and non-cancerous (N) areas of 99 cases of renal cancer patients, and each lectin signal was below the lower limit of the quantification range. are shown in italics. These lectins are omitted from FIGS. Combined with FIG. 21, there are 27 beneficial lectins with AUC (area under the curve)>0.8, among which lectins (16 species) are shown in bold. 21 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. FIG. Specifically, ROC analysis was performed using lectin array analysis data of cancerous (T) and non-cancerous (N) areas of 99 cases of renal cancer patients, and each lectin signal was below the lower limit of the quantification range. are shown in italics. These lectins are omitted from FIGS. Combined with FIG. 20, there are 27 beneficial lectins with AUC (area under the curve)>0.8, among which lectins (16 species) are shown in bold. 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. 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). 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. 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). 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). 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. (Right), and shows the results of the lectin group in which a particularly marked divergence occurred (significantly high value in non-cancerous area). In each boxplot, the distribution is shown by dividing into non-cancerous (left) and cancerous (right). 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. It is a figure which shows. A WFA signal-positive case was randomly selected from frozen tissue specimens of renal cancer patients, and ICAM-1 was immunoprecipitated (IP), treated with an enzyme, and added to a lectin array. In each graph, black (IP (ctrl)) is the result of adding the IP product as it is, white (IP Sialidase A) is the result of adding the IP product after sialidase A digestion, and diagonal lines (IP b14 Galactosidase) are the result of adding the IP product to β1,4 The results of addition after galactosidase digestion are shown, respectively. FIG. 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. In each graph, black (IP (ctrl)) is the result of adding the IP product as it is, white (IP Sialidase A) is the result of adding the IP product after sialidase A digestion, and diagonal lines (IP b14 Galactosidase) are the result of adding the IP product to β1,4 The results of addition after galactosidase digestion are shown, respectively. 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.

Hereinafter, embodiments for carrying out the present invention will be described based on the drawings. The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the invention, its application or its uses.

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. In addition, in this embodiment, the CLA is preferably a recombinant CLA.

The method according to the present embodiment, which will be described in detail in the examples below, 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. As shown in the examples below, among the above lectins, WFA, ECA, CLA, BPL, Discoidin II, RCA120, HHL, GNA, BC2LCN, KAA1, ESA2, MPA2, NPA, AAL and MPA1 are non-renal cancer The specificity for renal cancer tissue ICAM-1 is significantly higher than for tissue ICAM-1, while CGL2, WGA, STL, ACG, LEL, CFAsub1, LSL-N, CBA-pro, UEA-I and Jacalin has significantly lower specificity for renal cancer tissue ICAM-1 than for non-renal cancer tissue ICAM-1 (both AUC>0.8 in ROC analysis). Therefore, by evaluating the difference in reactivity between the lectin and ICAM-1 in a biological sample of the subject and ICAM-1 in a control non-cancerous tissue, the renal cancer of the subject is diagnosed or can be detected.

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.

In addition, 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. In addition, 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.

In this embodiment, the subject is a human or animal, including mice, guinea pigs, rats, monkeys, dogs, cats, hamsters, horses, cows and pigs, preferably humans.

In the present embodiment, 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. By doing so, even when the concentration of ICAM-1 in the biological sample is low, it is possible to quantify ICAM-1 with higher accuracy, which is preferable. The 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.

In this embodiment, 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. When the biological sample is renal tissue, for example, the renal tissue can be solubilized and the solution containing the lectin is added and mixed as shown in the examples below. In addition, 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.

In this embodiment, 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. Here, molecules that can specifically bind to ICAM-1 are not limited to antibodies, and peptides, aptamers, and the like may also be used. In the case of antibodies, monoclonal antibodies are preferred. The above quantification method is also not particularly limited, but for example, Western blotting using a labeled ICAM-1 antibody can be used, and other techniques commonly used as protein quantification means in this technical field can be used. . In particular, in conjunction with the step of contacting the lectin with the biological sample of interest, 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. In addition, for example, 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. can also

In the present embodiment, 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. When using an absolute value as a measurement value, for example, 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. At this time, 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. On the other hand, when using the above relative values as the measured values, 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 The relative values obtained for each of the lectins in are compared with each other, and if lectins with significantly different relative values are included, the subject may be diagnosed as having renal cancer.

Other embodiments of the present invention 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. Also in this embodiment, 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.

In this embodiment, 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. Although it is not limited to the following method as long as it is performed based on the comparison, for example, 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. When using an absolute value as a measurement value, for example, 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. At this time, 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. On the other hand, when using the above relative values as the measured values, 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.

Other embodiments of the present invention include CGL2, WFA, ECA, WGA, CLA, STL, BPL, Discoidin II, RCA120, ACG, LEL, CFAsub1, LSL-N, HHL, GNA, BC2LCN, CBA-pro, UEA- A diagnostic kit for kidney cancer, comprising at least one lectin selected from the group consisting of I, KAA1, ESA2, MPA2, NPA, AAL, Jacalin and MPA1, and a probe that specifically binds to ICAM-1 . In addition, in this embodiment, the CLA is preferably a recombinant CLA.

In the present embodiment, 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.

Examples are given below to describe in detail the method and kit for diagnosing or detecting renal cancer according to the present invention.

[Example 1: Exploratory experiment on changes in sugar chains on ICAM-1]
First, in order to preliminarily verify whether renal cancer-specific sugar chain changes occur on ICAM-1, we randomly selected 6 cases of renal cancer patients. Using the tissue, the following experiments were carried out.

(Enrichment of ICAM-1 in tissue specimens using anti-ICAM-1 antibody)
The cancerous (T) and peripheral non-cancerous (N) frozen tissues of the 6 cases of renal cancer patients ((1) to (6)) were solubilized according to a conventional method, and the obtained solution was used as a tissue lysate. bottom. The solubilization buffer used at that time was 1% Protease Inhibitor Cocktail, Animal Component Free, manufactured by Sigma-Aldrich)/1% Triton X-100-containing Phosphate-buffered saline (PBS) buffer. 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. As a result, 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)).

In the above immunoprecipitation, 10 μL of streptavidin-immobilized magnetic beads (Dynabeads MyOne Streptavidin T1, manufactured by DYNAL) used for preclearing, and 10 μL of the magnetic beads and biotinylated anti-ICAM-1 antibody (Human ICAM -1/CD54 Biotinylated Antibody (BAF720), manufactured by R&D Systems) 200 ng was used. A PBS buffer containing 1% Triton X-100 (PBSTx) was used to wash the beads. For elution of ICAM-1 captured by the magnetic beads, 10 μL of TBS buffer containing 0.2% SDS was added to the magnetic bead pellet after sedimentation, and heat treatment was performed at 95° C. for 10 minutes. The magnetic beads are trapped by a dedicated collection magnet, 20 μL of the magnetic beads are added to the obtained supernatant, and the biotinylated antibody eluted together with the relevant protein by heating is trapped and removed by the magnetic beads, and the final obtained supernatant is subjected to 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.

As shown in Figure 1, the presence of ICAM-1 was observed in both cancerous and non-cancerous areas in all six cases of renal cancer patients. The abundance ratio was significantly higher in the cancerous area, and interestingly, the mobility of ICAM-1 was different between the cancerous area and the non-cancerous area. Since such a phenomenon generally occurs when glycosylation such as sialic acid is different, it was strongly suggested that there is a difference in glycosylation between cancerous and non-cancer parts.

(Comparative glycan profiling of ICAM-1 in tissues by lectin array)
Next, in order to compare the types of lectins that recognize each of the sugar chains of ICAM-1 in cancerous and non-cancerous tissues, ICAM-1 enriched obtained by the above method was used, and Kuno et al. Lectin array analysis (antibody overlay method) was performed according to the method of Kuno A et al. 2020)). The lectin array chips used at that time were a commercially available chip LecChip ver1.0 and custom-manufactured ver2.0 (both manufactured by Glyco Technica). The lectins used in the lectin array chips ver1.0 and ver2.0 are shown in FIGS. 2 and 3, respectively. In addition, in the lectins shown in FIG. 3, those marked with "r" are recombinant, and those marked with "His" are those labeled with a His tag.

Each sample (ICAM-1-enriched) was applied to the lectin array chips ver1.0 and ver2.0 with the immunoprecipitated solution equivalent to 10 μg of total protein, and subjected to binding reaction at 20° C. for 10 hours or longer. . In order to prevent the sugar chains on the detection antibody from binding to unreacted lectin on the substrate and generating noise, 2 μL 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. Technica) was used to detect binding signals by array scanning. The results are shown in FIGS. In each graph of FIGS. 4 to 6, the vertical axis shows the relative signal value, and the horizontal axis shows 90 kinds of lectins contained in the lectin array chips ver1.0 and ver2.0. However, the order in which the lectins are arranged is the same in both graphs.

As shown in Figures 4 to 6, in cases (1) to (6), when comparing the results of the non-cancerous area in the upper row and the cancerous area in the lower row, it can be seen that the cancerous area and the non-cancer area It was found that the signal patterns differed at different sites, and some lectins showed an increase or decrease in signal at each site. A graph was created for the lectins for which P<0.05 was obtained by paired two-group comparison and the signal was significantly higher in the cancerous area (Tumor) than in the non-cancer area (Normal). , rMPA1, rMPA2, rESA2, AOL, AAL, CLA and ULL are shown in FIGS.

In summary, 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. Hereinafter, a group of lectins that preferentially bind to ICAM-1 present in cancerous areas will be referred to as cancerous specific lectins.

(Verification test of cancer specific lectin-1)
Next, in order to verify the adequacy of the cancer site-specific lectins selected by the lectin array analysis, a pull-down assay using rKAA1, one of the selected lectins, was performed. The method and results are described below. The rKAA1 used in the experiment was overexpressed in E. coli according to a conventional method, purified, and labeled with biotin. 10 μL of streptavidin-immobilized magnetic beads (Dynabeads MyOne Streptavidin T1, manufactured by DYNAL) used for preclearing. For lectin precipitation, 10 μL of the magnetic beads and 2 μg of biotin-labeled lectin were reacted to prepare lectin-immobilized magnetic beads and used. To this, pre-cleared tissue lysate equivalent to 20 μg of total protein was added, binding reaction was carried out, unbound substances were washed with PBSTx, and 10 μL 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. In addition to the target molecule ICAM-1, molecules that bind to KAA are also present in the rKAA-binding protein solution. To improve the purity of ICAM-1, 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.

As shown in FIG. 9, when compared with the case without rKAA1 collection (FIG. 1), in all of the 6 cases of renal cancer patients ((1) to (6)), it was clearly observed that after rKAA1 collection However, there is a significant difference in the intensity of ICAM-1 between cancerous (T) and non-cancerous (N) areas. That is, it can be seen that a significantly large amount of ICAM-1, which binds to rKAA1, is present in cancerous areas. From the above, it became possible to verify cancer specific lectins.

(Verification test of cancer specific lectin-2)
In order to verify whether the sugar chains assumed from the binding specificity of cancer-specific lectins are actually upregulated on ICAM-1 present in cancer, comparative sugar chain analysis by mass spectrometry was performed. rice field. ICAM-1 immunoprecipitated by the above method was analyzed to clarify what kind of sugar chain was added to which position on ICAM-1. For analysis, the glycoprotein analysis method established by Kaji et al. .org/10.1093/glycob/cwab060 (2021)). In addition, the frozen tissue lysate of the renal cancer patient (6) was used for the analysis. FIG. 10 shows the analysis results of N-type glycosylation to asparagine at position 406 of ICAM-1.

As shown in FIG. 10, some sugar chain structures that did not exist in the non-cancerous region (N) were generated in the cancerous region (T), and as shown in the table of FIG. There was a change in the abundance ratio (Relative%) of (High Man) and complex type sugar chains (Complex). In particular, several sugar chain structures that did not exist in the non-cancer site (N) were generated in the cancer site (T), and the Lewis formula fucose-containing double-chain complex-type sugar circled in the upper part of FIG. Chains (Lewis Fuc) and high mannose type sugar chains (Man5, Man7) were generated only in the cancer site (T). The former of these sugar chains is known to bind to AOL, AAL, CLA, etc. in FIG. 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]
(Lectin array analysis for multiple sample cases)
Next, 100 renal cancer patients with various clinical backgrounds were recruited to validate the exploratory study. The use of these specimens in this experiment has been approved by the ethics review of the Keio University School of Medicine. A total of 198 cancerous and non-cancerous tissue regions from 99 of these cases were subjected to comparative sugar chain analysis using a lectin array. The same method as in Example 1 was used for pretreatment of frozen tissue specimens, ICAM-1 enrichment, and lectin array analysis. In order to verify the qualitative changes of sugar chains on ICAM-1, the acquired signals in the lectin array analysis were normalized by the average value of each chip. Specifically, in this example, a value standardized by the average value of signals obtained from each of the 45 lectins on each lectin array chip is used. By calculating and comparing relative values using such a value that can be used as an internal standard, it is possible to verify qualitative changes rather than quantitative changes. Excluding 27 lectins that showed minor signals on the chip, box plots were created for 63 lectins. The results for lectin array chip ver1.0 are shown in FIGS. 15-18, respectively.

As shown in Figures 11 to 18, these results verified the validity of the above exploratory results. Interestingly, in this validation study, even though we recruited and analyzed patients with different conditions such as the degree of cancer progression, there was basically a clear difference between cancerous and non-cancerous areas. Multiple lectins were present. Since the difference is sometimes increased or decreased in cancerous areas, it is strongly suggested that it is not simply a change due to an increase or decrease in the expression of the ICAM-1 molecule, but a qualitative change in the sugar chain on the protein. .

(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.

In the clustering results shown in FIG. 19, cancerous areas represented by black lines and non-cancerous areas represented by white lines were well separated into separate clusters. They were shown to have different glycan profiles. In addition, since there were two cases of divergence seen in the center of FIG. However, when the state of expression of ICAM-1 was confirmed by Western blotting, it was found that the expression of ICAM-1 in these samples was significantly weaker than in other cancerous areas.

(Selection of cancer site-specific lectins by ROC analysis)
As mentioned above, we found that cancerous and non-cancerous areas can be clearly separated by examining the sugar chains on ICAM-1. In order to confirm whether or not there is any cancer, 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. In FIGS. 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. In addition, 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.

As shown in 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.

(Comparative glycan analysis using frozen tissue sample crude glycoprotein solution)
If cancer-specific changes in sugar chains on ICAM-1 occur in most of the glycoproteins in the cancerous part, similar changes should be detectable in the crude glycoprotein extract, making it easy to detect. becomes possible. In order to examine whether this hypothesis holds true, the frozen tissue lysate used in the ICAM-1 experiment was used as a crude glycoprotein solution, and the following comparative glycan analysis was performed according to the method of Wagatsuma et al. (Wagatsuma T et al. Frontiers in Oncology 10, 338 (2020)). 200 ng of protein-quantified tissue lysate was mixed with 10 μg of Cy3-SE (manufactured by GE Healthcare) to make 10 μL, reacted at room temperature for 60 minutes, and fluorescently labeled. In order to inactivate the functional groups of the excess labeling agent, 90 μL of probing buffer (TBSTx buffer containing 500 mM glycine/1% Triton X-100 (1 mM CaCl2, 1 mM MnCl2 is also included)) was added and incubated at room temperature for 2 hours. reacted. This is used as a fluorescence-labeled glycoprotein solution. The protein solution was added to the probing buffer so that the total protein amount was 15 ng equivalent to 60 μL, added to one well of the lectin array chip ver1.0, and reacted at 20° C. for 10 hours or more. . After the reaction, 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.

As shown in Figures 22 to 25, similar to the case of ICAM-1, which is a specific glycoprotein, the values were higher in cancerous areas than in non-cancer areas, no difference, and higher values in non-cancer areas. I was able to classify them into several patterns. In other words, it was found that cancer-associated changes in sugar chains also occur in a comprehensive comparative glycan analysis using a crude glycoprotein solution.

(Data comparison between crude glycoprotein and specific protein ICAM-1)
Before proceeding with the data comparison of the lectin array analysis of the crude glycoprotein solution of frozen tissue from 99 cases of renal cancer patients and the specific glycoprotein (ICAM-1) therein, we first tried to extract lectins with particularly marked discrepancies. . The results are shown in FIGS. 26-29.

Of the 45 lectins mounted on the lectin array chip ver1.0, 6 lectins (AOL, AAL, ECA, RCA120, BPL, WFA) shown in FIG. Although an increase was obtained, it could be classified into [1] a lectin in which no change was observed in a crude glycoprotein solution and [2] a lectin in which a decrease in signal was observed at cancerous sites. Interestingly, none of the four lectins (ECA, RCA120, BPL, WFA) classified in [1] have asialo-glycans, that is, do not have sialic acid modifications at the ends of N-glycans, and contain galactose to N-acetylgalactosamine. It recognized sugar chains in the exposed state. On the other hand, the two lectins (AOL, AAL) classified in [2] are fucose-recognizing lectins with low specificity (capable of recognizing various fucose modifications) and were selected in exploratory analysis. (See Figures 7 and 8). The discovery of cancer-specific change-recognizing lectins that cause such discrepancies cannot be achieved by the usual analysis using crude glycoprotein solutions alone. It was confirmed in this experiment that it can be achieved for the first time by performing

As another example of divergence, as shown in Fig. 27, 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)). However, none of the above four lectin signals, which are enhanced in cancer, was observed on Basigin in pancreatic cancer tissue.

In addition, according to this analysis, there was also a lectin that increased the signal at the cancer site in both ICAM-1 and the crude glycoprotein solution. There are four lectins (NPA, ConA, GNA, HHL) shown in FIG. 28, all of which are high-mannose sugar chain recognizing lectins. In addition, four lectins (ACG, LEL, STL, and WGA) that are commonly decreased in cancer sites were found (Fig. 29). It is interesting that it is a lectin that recognizes groups.

(Prediction of sugar chains on cancer-specific sugar chain-containing ICAM-1 by lectin array analysis of enzymatic digestion products)
Through a series of analyses, a group of galactose/N-acetylgalactosamine-recognizing lectins (ECA, RCA120, BPL, and WFA for lectin array chip ver1.0), which are expected to have high results in both sensitivity and specificity, in the diagnosis of renal cancer. rLSL-N, rDiscoidinI, rDiscoidinII, etc. in version 2.0) actually recognized sugar chains on ICAM-1. Lectin array analysis of isolated ICAM-1 enzymatic digests was performed. Note that many of these lectins do not allow modification of sialic acid. Therefore, when an increase in signal is confirmed by digestion with sialidase A, which is a sialic acid-degrading enzyme, it is evidence of terminal sialic acid modification. Also, many of these lectins recognize galactose to N-acetylgalactosamine. In addition, it recognizes β1,3-1,4-linked Gal as the binding position. Therefore, this time, we decided to perform β-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 β1,4 galactose at the end.

Among the frozen tissue specimens of 100 cases of renal cancer patients, one positive case and one negative case for the WFA signal of the galactose/N-acetylgalactosamine-recognizing lectin are randomly selected and used in this experiment. made it Immunoprecipitation (IP) and elution/isolation of ICAM-1 from the frozen tissue lysate solution were performed by the above method. Each enzyme was then added, treated at appropriate temperature and time, and the reaction product was added to the lectin array. At that time, a buffer solution was added instead of the enzyme, and the reacted sample was used as a control sample. The results are shown in FIGS. 30 and 31. FIG.

As shown in FIGS. 30 and 31, in the positive case (FIG. 30), the signal of sialic acid-recognizing lectins (MAL-I, SNA, SSA, TJAI) is relatively weak in the untreated state (black), and the asialosaccharide Chain recognition lectins (ECA, RCA120, BPL, WFA) were relatively high. After sialic acid cleavage (white), the lectin recognizing sialic acid decreased in response, but the lectin recognizing asialo-glycans did not change significantly. When β1,4-galactosidase digestion was performed while sialic acid was retained, the asialo-glycan-recognizing lectin lost its signal. Therefore, it was found that ICAM-1 in this case has very little terminal sialic acid modification and β1,4Gal is exposed. On the other hand, in the negative case (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 α2,6 sialic acid modification of Gal and can bind to it. After the cleavage of sialic acid (white), the reaction of the lectin recognizing sialic acid disappeared, but it was confirmed that the reaction of the lectin recognizing asialo-glycans increased markedly compared to the case of the above-mentioned positive cases. When β1,4-galactosidase digestion was performed while sialic acid was retained, there was no change in BPL and WFA, since there was no reaction from the beginning, and the signals in ECA and RCA120, which showed signals in the untreated state, decreased. In summary, it was found that ICAM-1 in this case has a large number of terminal sialic acid modifications, and most of the modified galactose is β1,4 linkage.

[Example 3: Evaluation of cancer detectability by combination of two lectins]
In Example 2 above, as described above, in order to verify the qualitative changes in the sugar chains of ICAM-I, the values standardized by the average value of all lectin signals on the lectin array chip were used. In the example, it was tested whether cancer could be detected based on the absolute value of each lectin's signal without such standardization. That is, the absolute values of the signals of the two types of lectins in each of the cancerous and non-cancerous areas were directly used for evaluation. Specifically, the signal data of the lectin array (LecChip ver1.0 and 2.0) obtained in Example 2 were used as they were. As described above, an example of a lectin having different specificity for four of the asialo-glycan recognizing lectins, WFA, RCA120, ECA and DiscoidinII, is shown in FIGS. Two-dimensional plots were drawn using CLA, the lectin with the highest T and highest AUC values.

As shown in Figures 32 and 33, two-dimensional plots of the absolute values of the signals of CLA and asialo-glycan-recognizing lectins (RCA120, Discoidin II or ECA) show that the signals of these lectins are small in non-tumor areas (white). Concentrate on the lower left area. On the other hand, in cancerous areas (black), the signal values for all lectins were several times higher than those in non-cancer areas in most cases, and the two could be clearly distinguished.

From the results of this example, using one or more cancer-specific lectins, the quantitative value of the lectin bound to ICAM-1 in the non-cancer part as a control and the ICAM-1 in the cancer part It was suggested that kidney cancer can be detected based on the difference from the quantified value of the bound lectin.

As described above, it has been clarified that 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.

Claims (7)

A method for diagnosing kidney cancer, comprising:
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 taken from the 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 for detecting kidney cancer, comprising:
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 taken from the subject;
quantifying ICAM-1 bound to said lectin in said biological sample;
and determining whether or not renal cancer is detected in the subject based on the comparison of the amount of ICAM-1 bound to the lectin and a predetermined reference value. The method according to claim 1 or 2, wherein the biological sample is renal tissue, blood or urine. The method according to any one of claims 1 to 3, wherein the step of quantifying ICAM-1 is performed using a probe that specifically binds to said ICAM-1. The method according to any one of claims 1 to 4, further comprising the step of concentrating said biological sample prior to contacting said lectin with said biological sample. A diagnostic kit for renal cancer,
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 at least one lectin selected from the group consisting of MPA1;
a probe that specifically binds to ICAM-1. 7. The kit of claim 6, wherein said probe comprises an antibody, peptide or aptamer that specifically binds to ICAM-1.

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