Method for determining onset risk of hepatocellular carcinoma in hepatitis c virus-infected subject having achieved virological response, and method for screening for compound that reduces said risk

Abstract

Provided are: a method for determining the onset risk of hepatocellular carcinoma in a hepatitis C virus-infected subject having achieved virological response, the method being characterized by measuring binding between a Mac-2 binding protein and at least two types of lectin selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and a method for screening for a compound that reduces said risk.

Description

Method for determining the risk of developing hepatocellular carcinoma in subjects infected with hepatitis C virus who have achieved a complete virological response, and method for screening for compounds that reduce said risk

[0001] The present invention relates to a method for determining the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a virological response, and a method for screening for a compound that reduces the risk.

[0002] Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths, with approximately 870,000 new cases and 760,000 deaths expected in 2022. Hepatocellular carcinoma (HCC), the most common type of liver cancer, is primarily caused by chronic hepatitis C virus (HCV) infection. If untreated, 50% of patients with cirrhosis due to chronic HCV infection will develop HCC within 10 years. Meanwhile, recently launched direct-acting antivirals (DAAs) are highly effective in treating HCV infection, achieving a nearly 100% sustained virologic response (SVR) with only 12 weeks of oral administration. While this has significantly reduced the risk of developing HCC, even in SVR cases, approximately 4% of patients develop cancer within five years.

[0003] Early detection of cancer is important in cancer treatment. In the case of HCC, identifying its onset early is also important for ensuring a good prognosis. To detect HCC early, imaging tests such as ultrasound, MRI, and CT scans, as well as blood tests to measure HCC markers, are used. However, currently used HCC markers, such as alpha-fetoprotein (AFP), AFP-L3, and vitamin K deficiency protein II (PIVKA-II), lack sensitivity and performance for early detection.

[0004] A glycosylated isomer (M2BPGi) of Mac-2 binding protein (M2BP), recognized by Wisteria floribunda agglutinin (WFA), has attracted attention as a predictive marker for HCC (Patent Document 1, Non-Patent Document 1). The application of M2BPGi to identify and follow-up patients at high risk for HCC in cases where SVR was achieved after DAA treatment is expected. However, because M2BPGi levels decrease in all cases after DAA treatment, it is difficult to determine a clear cutoff value. Therefore, a method for early assessment of HCC risk, independent of DAA treatment, is needed.

[0005] International Publication No. 2011 / 007797

[0006] Yamasaki, K., et al., Hepatology, 2014; 60(5):1563-1570

[0007] The present invention has been made with the objective of providing a method capable of determining with high accuracy the risk of developing HCC in HCV patients who have achieved SVR.

[0008] As a result of extensive research, the present inventors found that the glycosylation signature of M2BP after DAA treatment differed between patients who developed HCC after achieving SVR and those who did not.

[0009] That is, according to one embodiment, the present invention provides a method for determining the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a virological response, the method comprising: (1) contacting a Mac-2 binding protein in a sample derived from the subject with at least two types of lectins, wherein the lectins are selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and (2) measuring the binding of each of the at least two types of lectins to the Mac-2 binding protein.

[0010] The at least two types of lectins are preferably selected from the following (i) to (vi): (i) TJA-I or SNA, (ii) LCA or PSA, (iii) AAL, (iv) ECA, (v) AOL, and (vi) WFA.

[0011] The at least two types of lectins preferably include AOL and / or WFA.

[0012] Alternatively, the at least two types of lectins preferably include LCA.

[0013] Preferably, the above method further comprises the step of: (3) normalizing the measurement values ​​for each of the at least two types of lectins obtained in step (2) by the average of the measurement values ​​for all of the at least two types of lectins.

[0014] Preferably, the sample is blood.

[0015] The method may further comprise, before step (1), a step of capturing a Mac-2 binding protein in a sample derived from the subject.

[0016] In another embodiment, the present invention provides a method for screening for a compound that reduces the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a complete virological response, the method comprising the steps of: (1) contacting hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts derived from hepatocellular carcinoma tissue with a candidate compound; (2) capturing a Mac-2 binding protein derived from the hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts; (3) contacting the Mac-2 binding protein with at least two lectins, wherein the lectins are selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and (4) measuring binding of each of the at least two lectins to the Mac-2 binding protein.

[0017] The at least two types of lectins are preferably selected from the following (i) to (vi): (i) TJA-I or SNA, (ii) LCA or PSA, (iii) AAL, (iv) ECA, (v) AOL, and (vi) WFA.

[0018] The at least two types of lectins preferably include AOL and / or WFA.

[0019] Preferably, the above method further comprises the step of: (5) normalizing the measured values ​​for each of the at least two types of lectins obtained in step (4) by the average of the measured values ​​for all of the at least two types of lectins.

[0020] The method of the present invention allows for the detection of M2BP glycosylation signatures specific to patients who will develop HCC after achieving SVR using only a few lectins, making it possible to easily and early identify patients at high risk of developing HCC after achieving SVR.

[0021] Furthermore, according to the screening method of the present invention, compounds that change the glycosylation signature of M2BP specific to patients who develop HCC after achieving SVR can be obtained as candidate compounds for drugs that reduce the risk of developing HCC.

[0022] Figure 1 shows the results of Western blotting confirming M2BP expression in various HCC cell lines. Figure 2 shows the results of principal component analysis using lectin array data for M2BP in the culture supernatants of various HCC cell lines and the serum of healthy individuals. Figure 3 shows the results of principal component analysis using lectin array data for M2BP in the serum of patients who developed HCC after DAA treatment and patients who did not. Figure 4 shows a graph comparing the signals of six lectins in fully automated lectin array analysis of M2BP in the serum from one patient (randomly selected) who developed HCC after DAA treatment, before DAA treatment, immediately after treatment, SVR12, SVR24, SVR48, and at the time of HCC observation. Figure 5 shows the results of principal component analysis using lectin array data for the serum (without M2BP capture treatment) of patients who developed HCC after DAA treatment and patients who did not develop HCC. Figure 6 is a graph showing the LCA, AOL, and WFA signals in the serum of an HCC group and a control group (10 cases each, selected at random) measured using a multilectin bead array chip packed with a total of 12 types of beads, including 11 types of lectin beads and anti-M2BP antibody beads. Figure 7 is a graph showing the LCA, AOL, and WFA signals in the serum of an HCC group and a control group (10 cases each, selected at random) measured using an LCA bead-packed chip, an AOL bead-packed chip, and a WFA bead-packed chip.

[0023] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0024] According to a first embodiment, the present invention provides a method for determining the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a virological response, the method comprising: (1) contacting a Mac-2 binding protein in a sample derived from the subject with at least two types of lectins, wherein the lectins are selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and (2) measuring binding of each of the at least two types of lectins to the Mac-2 binding protein.

[0025] "Hepatocellular carcinoma (HCC)" refers to a type of primary liver cancer in which liver cells become cancerous. Cholangiocarcinoma, which develops in the bile duct, is not included in hepatocellular carcinoma. Hepatocellular carcinoma is strongly associated with viral infection, progressing from cirrhosis caused by persistent infection with hepatitis viruses to hepatocellular carcinoma. There are five main types of hepatitis viruses: A, B, C, D, and E, and persistent infection with hepatitis B virus (HBV) and especially hepatitis C virus (HCV) causes hepatocellular carcinoma.

[0026] The "subject" in this embodiment may be any animal that can be infected with HCV, preferably a primate, and particularly preferably a human.

[0027] In this embodiment, the "risk of developing hepatocellular carcinoma" refers to the possibility that a subject will develop hepatocellular carcinoma, specifically, the possibility that HCC cells will appear and / or proliferate in a subject.

[0028] "Severe Virologic Response (SVR)" refers to the complete elimination of the virus from the body of an infected subject. Generally, SVR is defined by undetectable viral RNA in the blood 12 or 24 weeks after completion of antiviral treatment (referred to as SVR12 or SVR24, respectively). The SVR in this embodiment may be SVR12, SVR24, or higher (e.g., SVR48). Anti-HCV treatments capable of achieving SVR have already been established, and for example, administration of direct-acting antivirals (DAAs) such as ledipasvir / sofosbuvir can achieve SVR in nearly 100% of cases.

[0029] In the method of this embodiment, a Mac-2 binding protein in a sample derived from a subject is contacted with at least two types of lectins.

[0030] The "sample" in this embodiment may be a biopsy tissue or body fluid from a subject, but is preferably a body fluid, and particularly preferably blood, plasma or serum.

[0031] Mac-2 binding protein (M2BP) (also known as galectin-3 binding protein or 90K) is a highly glycosylated secreted glycoprotein. The M2BP glycosylation isomer (M2BPGi), recognized by WFA (Wisteria floribunda agglutinin (lectin)), is known as a liver fibrosis marker. A method for assessing liver fibrosis based on M2BPGi has already been well established (e.g., International Publication No. 2011 / 007797), and kits for this purpose are commercially available (e.g., HISCL M2BPGi Reagent: Sysmex Co.).

[0032] "Lectin" is a general term for proteins other than antibodies that recognize and bind to glycans, and they exist in a wide range of organisms, from animals to plants, fungi, and viruses. In the method of this embodiment, a lectin selected from the group consisting of the following 17 types of lectins is used: a lectin derived from Trichosanthes japonica (TJA-I), a lectin derived from Aleuria aurantia (AAL), a lectin derived from Erythrina cristagalli (ECA), a lectin derived from Lens culinaris (LCA), a lectin derived from Aspergillus oryzae (AOL), a lectin derived from Pisum sativum (PSA), a lectin derived from Sambucus nigra (SNA), a lectin derived from Griffonia nigra (Griffonia nigra ...), a lectin Lectin from Narcissus pseudonarcissus (NPA), jack bean (Canavalia ensiformis) (ConA), willow mushroom (Agrocybe cylindracea) (ACG), purple melon (Bauhinia purpurea alba) (BPL), Trichosanthes japonica (Trichosanthes japonica) (TJA-II), Agaricus bisporus (ABA), and nettle (Urtica dioica (UDA), Datura stramonium (DSA) and Wisteria floribunda (Wisteria floribunda) lectin (WFA).

[0033] In the method of this embodiment, at least two different lectins arbitrarily selected from the above are used in combination, but preferably three or more, more preferably four or more, and particularly preferably five or more different lectins can be used in combination. In this case, it is more preferable to use a combination of lectins with different sugar chain specificities, and any one of the lectins with overlapping sugar chain specificities can be selected. For example, PSA for LCA, TJA-II for BPL, and SNA for TJA-I have overlapping sugar chain specificities, and any one of these can be selected.

[0034] Therefore, in the method of this embodiment, it is preferable to use a combination of two or more lectins selected from (i) either TJA-I or SNA, (ii) either LCA or PSA, (iii) AAL, (iv) ECA, (v) AOL, and (vi) WFA. Particularly preferably, a combination of two or more lectins including AOL and / or WFA, or a combination of two or more lectins including LCA may be used. Specifically, but not limited to, combinations such as LCA and AOL; LCA and WFA; AOL and WFA; TJA-I, AAL and WFA; TJA-I, AAL and ECA; TJA-I, LCA and AAL; AAL, AOL and ECA; AAL, ECA and WFA can be used, and these combinations can be further combined with one or more lectins selected from the above.

[0035] Lectins may be prepared by any method known in the art. For example, they may be purified from the organisms from which they originate, or nucleic acids encoding the lectins may be prepared by genetic engineering methods and introduced into host cells such as Escherichia coli for expression. Information on the amino acid sequences of lectins and the nucleic acid sequences encoding them can be obtained from a predetermined database via a portal site such as the GlyCosmos Portal (https: / / glycosmos.org / ). The above-mentioned lectins are also commercially available, and they may be used in the method of this embodiment.

[0036] The method of this embodiment may include a step of capturing M2BP in a sample derived from a subject prior to contacting the M2BP in the sample with a lectin. M2BP can be captured using established procedures. Specifically, M2BP can be captured by contacting a sample derived from a subject with an anti-M2BP antibody immobilized on a support, such as a plate or beads. The captured M2BP can be used in the next step after elution from the support or while still bound to the support. The anti-M2BP antibody that can be used in the method of this embodiment is not particularly limited as long as it can specifically recognize the protein portion of M2BP. It may be either a polyclonal or monoclonal antibody, or an antigen-binding fragment with equivalent recognition ability, such as Fab, F(ab')2, or scFv. Such anti-M2BP antibodies are commercially available, and these may also be used.

[0037] In some embodiments, either or both of M2BP and lectin captured from a sample derived from a subject may be immobilized on a solid support. The solid support may be, for example, based on an inorganic material such as glass, or a polymeric material such as polystyrene or polyethylene terephthalate, and may be appropriately selected depending on the measurement technique. The solid support may have any shape suitable for the measurement system, such as a membrane, beads, a multiwell plate, or a multi-array chip. Immobilization can be performed using established general techniques. For example, the lectin or M2BP may be directly coupled to the solid support surface via a covalent bond, captured by an anti-lectin antibody or anti-M2BP antibody immobilized on the solid support, or indirectly coupled via avidin-biotin or the like. Lectin-immobilized arrays are commercially available, and such commercially available products can also be used in the method of this embodiment. For example, LecChip (Precision System Science) is a preferred commercially available product.

[0038] In some embodiments, a detectable label may be introduced into the M2BP or lectin captured from a sample derived from a subject. Detectable labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, metal particles, etc. The detectable label may be introduced into the lectin or M2BP directly or indirectly via an antibody, etc., according to any method known in the art.

[0039] Contact between M2BP and lectin can be achieved by incubating a sample derived from a subject or captured M2BP with the lectin for a certain period of time in a physiological buffer such as phosphate-buffered saline or Tris-HCl buffer. The concentrations of M2BP and lectin and the incubation time may be appropriately determined depending on the method used to measure the binding between them. For example, in the case of a lectin array, the M2BP concentration may be, for example, 10 ng / mL to 1 μg / mL, and the incubation time may be, for example, 1 minute to 24 hours.

[0040] Next, the binding between the lectin and M2BP is measured. The binding between the lectin and M2BP can be measured by any technique for analyzing intermolecular interactions. Examples of such techniques include, but are not limited to, lectin-antibody sandwich ELISA, lectin array, high-performance liquid chromatography mass spectrometry, capillary electrophoresis mass spectrometry, surface plasmon resonance (SPR), and fluorescence resonance energy transfer (FRET). In this embodiment, a lectin array is preferably used, and an antibody-overlay lectin array is particularly preferably used. Examples of lectin arrays and antibody-overlay lectin arrays include lectin microarrays, lectin barcode arrays, and lectin bead arrays.

[0041] According to the method of this embodiment, the risk of HCC in a subject can be determined based on whether or not an M2BP glycosylation signature correlates with the risk of HCC. In patients who develop HCC after achieving SVR, the M2BP glycosylation signature remains unchanged before and after anti-HCV treatment, whereas in patients who do not develop HCC after achieving SVR, the M2BP glycosylation signature changes after anti-HCV treatment. Therefore, the method of this embodiment may further include a step of comparing the M2BP glycosylation signature in the analytical sample obtained as described above with the M2BP glycosylation signature in a reference sample collected from a patient (control) who did not develop HCC after achieving SVR. Alternatively, the method of this embodiment may further comprise a step of comparing the glycosylation signature of M2BP in the analytical sample obtained as described above with the glycosylation signature of M2BP in a reference sample collected from the same subject in advance (e.g., before, during, or immediately after anti-HCV treatment). The glycosylation signature of the reference sample may be obtained in parallel with the analytical sample, or may be prepared separately in advance. If a significant difference is observed between the measured values ​​for the sample derived from the subject and the measured values ​​for the reference sample, it can be determined that the subject may be at risk of developing HCC.

[0042] When comparing measured values, it is preferable to normalize the measured values. In the method of this embodiment, for example, the measured values ​​for each of the at least two types of lectins used in the measurement may be normalized by the average of the measured values ​​for all of the at least two types of lectins. This is preferable because it does not require measurement of additional lectins for normalization. Specifically, for example, in a specific embodiment using TJA-I, AAL, and ECA, the measured values ​​of TJA-I, AAL, and ECA may be normalized by the average of all the measured values ​​of TJA-I, AAL, and ECA. In a specific embodiment using TJA-I, LCA, AAL, and ECA, the measured values ​​of TJA-I, LCA, AAL, and ECA may be normalized by the average of all the measured values ​​of TJA-I, AAL, and ECA. In the method of this embodiment, normalization using the measured values ​​for an additional lectin and / or anti-M2BP antibody may be performed instead of or in addition to the above normalization.

[0043] The measurement results may be evaluated using known statistical analysis methods. For example, the measurement results may be subjected to dimensionality reduction using multivariate analysis such as principal component analysis (PCA) or t-distributed stochastic neighbor embedding (t-SNE), and an index representing the glycosylation signature of M2BP may be derived. Furthermore, the glycosylation signatures in the analytical sample and the reference sample may be compared using known statistical analysis methods. For example, the glycosylation signatures in the analytical sample and the reference sample can be compared based on p-values ​​calculated by univariate analysis such as Student's t-test, and the accuracy of the determination (sensitivity and specificity) can be evaluated based on the ROC curve and AUC. Software such as JMP (registered trademark) can be used for statistical analysis.

[0044] According to a second embodiment, the present invention provides a method for screening for a compound that reduces the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a complete virological response, the method comprising the steps of: (1) contacting hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts derived from hepatocellular carcinoma tissue with a candidate compound; (2) capturing a Mac-2 binding protein derived from the hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts; (3) contacting the Mac-2 binding protein with at least two types of lectin, wherein the lectin is selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and (4) measuring binding of each of the at least two types of lectin to the Mac-2 binding protein.

[0045] In this embodiment, "hepatocellular carcinoma (HCC)," "subject," "risk of developing hepatocellular carcinoma," "severe virological response (SVR)," "Mac-2 binding protein (M2BP)," and "lectin" are the same as those defined in the first embodiment.

[0046] In the screening method of this embodiment, a candidate compound is contacted with hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts derived from HCC tissue. Hepatocytes, hepatic stellate cells, and hepatic fibroblasts are all known to secrete M2BP and can be used in the screening method of this embodiment. The hepatocytes, hepatic stellate cells, and hepatic fibroblasts in this embodiment may be primary cultures of cells isolated from the liver tissue of an HCC patient, or may be established cell lines. Examples of cell lines that can be used in this embodiment include HAK1A, HAK1B, HLF, PLC / PRF / 5, Li-7, SK-HEP-1, JHH-1, JHH-4, JHH-5, JHH-7, SNU-387, SNU-423, SNU-475, SNU-761, SNU-878, and HepG2. In the screening method of this embodiment, any of the above cell types may be used alone or in combination. Hereinafter, hepatocytes, hepatic stellate cells and / or hepatic fibroblasts derived from HCC tissue are referred to as "cells derived from HCC tissue."

[0047] In the screening method of this embodiment, liver spheroid or organoid can be prepared from HCC tissue-derived cells.The method for preparing liver spheroid or organoid has already been established, and for example, can be prepared liver spheroid or organoid by cell non-contact culture method, hanging droplet method, rotary culture method, three-dimensional culture method etc.The kit for preparing liver spheroid or organoid is also commercially available, and can be used for this commercially available product.

[0048] The liver spheroid or organoid in this embodiment can be used in vitro or in vivo.In other words, the screening method of this embodiment can be used in the animal model (for example, mouse or rat model) that is transplanted with the liver spheroid or organoid prepared from HCC tissue-derived cells.Such animal model includes, for example, LEC rat, high-fat diet NASH mouse, human liver chimeric mouse etc.

[0049] The "candidate compound" in this embodiment may be a low molecular weight compound, a nucleic acid, a protein, a peptide, an antibody, a lipid, or a mixture thereof (for example, an extract from animal or plant cells or tissues, a fungus or its homogenate or extract, or a culture supernatant thereof). Furthermore, these candidate compounds may be novel or known.

[0050] To contact a candidate compound with HCC tissue-derived cells in vitro, for example, the candidate compound may be added to a culture medium for culturing HCC tissue-derived cells or a physiological buffer solution such as phosphate-buffered saline or Tris-HCl buffer, and the cells may be incubated therein for a certain period of time. The final concentration of the candidate compound varies depending on the type of candidate compound, but for example, for low molecular weight compounds, it can be appropriately selected in the range of 1 pM to 1 μM. The incubation time may be, for example, 4 hours to 96 hours.

[0051] To contact a candidate compound with HCC tissue-derived cells in vivo, the candidate compound may be administered to an HCC animal model, for example, by intravenous, subcutaneous, intradermal, intralymph node, intraperitoneal, or intratumoral injection. The dosage may vary depending on the age, body weight, health condition, etc. of the subject, but may be, for example, 0.1 mg to 500 mg / kg (body weight).

[0052] Next, M2BP derived from HCC tissue-derived cells (hereinafter referred to as "HCC-derived M2BP") is captured from the medium or buffer solution after the incubation using, for example, an anti-M2BP antibody, in the same manner as in the first embodiment for capturing M2BP in the sample.

[0053] Next, the HCC-derived M2BP is contacted with at least two types of lectins. As in the first embodiment, the at least two types of lectins used in the screening method of this embodiment are selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, and WFA. Preferred combinations of lectins may be the same as in the first embodiment.

[0054] Next, the binding between the lectin and HCC-derived M2BP is measured. The measurement in this embodiment may be carried out in the same manner as in the first embodiment.

[0055] To determine whether the addition of a candidate compound has changed the amount of HCC-derived M2BP, a similar analysis can be performed in parallel without the addition of the candidate compound and compared, or the results can be compared with the results of a previous analysis performed without the addition of the candidate compound. In the method of this embodiment, if the amount of HCC-derived M2BP is significantly reduced by the addition of a candidate compound, the candidate compound can be determined to be a promising compound for reducing the risk of HCC development in HCV-infected subjects who have achieved SVR. On the other hand, if the amount of HCC-derived M2BP is the same as or increased compared to the amount without the addition of the candidate compound, the candidate compound can be determined to be unpromising as a compound for reducing the risk of HCC development in HCV-infected subjects who have achieved SVR.

[0056] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0057] <1. Glycosylation profiling of M2BP expressed in HCC cell lines> (1-1) Cell lines Six hepatocellular carcinoma (HCC) cell lines, HAK1A, HAK1B, HLF, KYN1, HepG2, and HuH7, were obtained from RIKEN or the JCRB Cell Bank. The HCC markers alpha-fetoprotein (AFP) and AFP-L3 were positive in HepG2 and HuH7, but negative in HAK1A, HAK1B, HLF, and KYN1.

[0058] HLF, HepG2, and HuH7 were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific Inc.) or RPMI 1640 medium (Thermo Fisher Scientific Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics under 5% CO ２ HAK1A, HAK1B, and KYN1 were cultured in RPMI 1640 medium (Thermo Fisher Scientific Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics under 5% CO ２ The cells were cultured at 37°C in a 1000-well incubator. After reaching 60-80% confluence, they were washed three times with Dulbecco's phosphate-buffered saline (D-PBS) and cultured for an additional 48 hours in DMEM or RPMI 1640 medium without FBS or antibiotics. The culture supernatant was then collected, filtered through a 0.45-µm disk filter, and used in subsequent experiments.

[0059] (1-2) Concentration of M2BP in the culture supernatant: Immunoprecipitation was performed according to the method of Kuno et al. (Kuno A. et al., Molecular & Cellular Proteomics, 8(1):99-108(2009)). Anti-human M2BP polyclonal antibody (R&D Systems, Inc.) was purified using MonoSpinProG (GL Sciences Inc.) and biotinylated using the biotin labeling kit NH2 (Dojindo Laboratories). The culture supernatant (1 μL) prepared in (1-1) above and biotinylated anti-M2BP antibody (100 ng) were mixed in 45 μL of Tris-buffered saline (TBSTx) containing 1% Triton X-100 and shaken at 4°C for 30 minutes. Streptavidin-conjugated magnetic beads (100 μg) were then added, and the mixture was shaken for an additional 30 minutes. After washing the beads three times with TBSTx, 18 μL of 0.1 M glycine-HCl (pH 2.7) containing 1% Triton X-100 was added and the mixture was shaken for 5 minutes to elute the molecules bound to the beads. The eluate was neutralized by adding 1 / 10 volume of 1 M Tris-HCl (pH 9.0), and this was used as the enriched M2BP solution in subsequent experiments.

[0060] (1-3) Western Blotting: One-fifth of the resulting enriched M2BP solution was electrophoresed on a 5-20% polyacrylamide gel under reducing conditions and transferred to a PVDF membrane. The membrane was blocked with BlockAce (Bio-Rad Laboratories, Inc.) and then incubated with 0.15 μg / mL biotinylated anti-M2BP antibody, followed by incubation with HRP-conjugated streptavidin (1 / 100,000 dilution, Jackson ImmunoResearch Laboratories, Inc.). Bands were detected using ImmunoStarLD (Fujifilm Wako Pure Chemical Industries, Ltd.), and band intensity was quantified using ImageJ software.

[0061] The results are shown in Figure 1. All six HCC cell lines (a: HAK1A, b: HAK1B, c: HLF, d: KYN1, e: HepG2, and f: HuH7) were confirmed to express M2BP at various levels.

[0062] (1-4) Glycosylation profiling of M2BP by lectin array Next, lectin array analysis (antibody overlay method) was performed according to the method of Kuno et al. (Kuno A. et al., Molecular & Cellular Proteomics, 8(1):99-108(2009)). An appropriate amount of the enriched M2BP solution was diluted with 60 μL of PBS solution containing 1% Triton X-100 (PBSTx) and subjected to lectin microarray (LecChip 45-uni, Precision System Science Co., Ltd.). After overnight incubation at 20°C, 20 μg of human serum polyclonal IgG was added to the chip as a blocking agent and incubated for 30 minutes. After washing three times with PBSTx, 100 ng of biotinylated anti-M2BP antibody was added to 60 μL of PBSTx and incubated for 60 minutes. After washing three times with PBSTx, 200 ng of Cy3-labeled streptavidin (GE Healthcare Technologies Inc.) was added to 60 μL of PBSTx and incubated for 30 minutes. After washing three more times with PBSTx, the glass slides were scanned using a dedicated array scanner (Glycostation Reader 2300, GlycoTechnica Ltd.). The scanned data were digitized and quantified using GlycoStation Tools Pro version 1.5 (GlycoTechnica Ltd.), and the average signal intensity of the three spots, after subtracting the background intensity, was used as the net signal intensity in the following process. The signal for each lectin was expressed as a value normalized by the average net signal of the 45 lectins. Principal component analysis was performed using the data using R software version 4.3.2. For the data from the lectin microarray analysis, no scaling between lectin signals was performed, and the normalized average value was used. The 45 lectins used in the lectin array are listed in Table 1.

[0063] Table 1. Lectins used in the lectin array

[0064] The results of principal component analysis are shown in Figure 2. The left panel shows plots of six HCC cell lines (a: HAK1A, b: HAK1B, c: HLF, d: KYN1, e: HepG2, and f: HuH7) and healthy donor serum (N), while the right panel shows plots of the 45 lectins. Principal component 1 enabled separation of AFP-positive HCC cell lines (HepG2 and HuH7) from AFP-negative HCC cell lines (HAK1A, HAK1B, HLF, and KYN1). The signals of α2,6-sialic acid-recognizing lectins (SNA, SSA, and TJA-I) were significantly higher in the AFP-positive HepG2 and HuH7 cells and healthy donor serum. On the other hand, AFP-negative HAK1A, HAK1B, and HLF cells showed relatively significantly higher signals from asialoglycan-recognizing lectins (ECA, RCA120, BPL, TJA-II, and WFA). Therefore, we measured M2BP glycosylation isomer (M2BPGi) levels based on WFA reactivity, and found that strong signals were observed only in AFP-negative HCC cell lines (data not shown).

[0065] These results demonstrate that M2BP is expressed in all HCC cell lines, whereas WFA-binding M2BPGi is expressed only in AFP-negative HCC cell lines, revealing differences in the amount of M2BP glycosylation isomers. Interestingly, M2BP expressed in AFP-positive HCC cells was not reactive to WFA but was reactive to α2,6 sialic acid-recognizing lectins, and was found to be similar to M2BP contained in the blood of healthy individuals. These results suggest that quantifying the changes in the glycosylation status of M2BP (glycosylation signature) is more useful than simply examining the amount of the glycosylation isomers.

[0066] 2. Glycosylation Profiling of M2BP in Serum from HCV Patients after DAA Treatment (2-1) Serum Sample Collection. Serum samples from HCV patients were collected from three different clinical institutions. The subjects of this analysis were HCV patients who achieved a complete virologic response (SVR) with direct-acting antivirals (DAAs) between January 2015 and December 2023. SVR was defined as the absence of detectable HCV RNA for an extended period after DAA treatment. Regular follow-up assessments, including tumor biomarkers (PIVKA-II and AFP) and imaging scans, were performed at 6- to 12-month intervals. Patients with a history of HCC or ultrasound-detected HCC at enrollment, severe inflammatory disease, or malignancies unrelated to the study focus were excluded.

[0067] (2-2) Study Design: This study identified the relationship between six parameters potentially associated with the development of HCC (AST, ALT, PLT, ALB, AFP, and AFP-L3%) and M2BPGi. Patients who developed HCC during the follow-up period were defined as the HCC group. Patients who did not develop HCC during the follow-up period were defined as the control group. At baseline, control patients were matched with HCC patients with specific pathological conditions using propensity score matching, including age, sex, ALT, PLT, AFP-L3%, and M2BPGi-Qt. AFP and AFP-L3 levels were measured using a μTAS Wako i30 fully automated immunoassay system (Fujifilm Wako Pure Chemical Industries, Ltd.). M2BPGi-Qt levels were measured using a fully automated HISCL-5000 immunoassay kit (Sysmex Co.). AFP-L3% was calculated as the ratio of AFP-L3 to AFP, and AFP-L3 below the lower limit of detection was defined as 0%.

[0068] (2-3) Clinical Characteristics of Serum Samples at Baseline A total of 910 patients were observed for a median of 1,542 days. During this period, 42 patients were diagnosed with HCC and classified into the HCC group. After propensity score matching for patients who did not develop HCC during the observation period, the control group consisted of 43 patients. As shown in Table 2, there were no significant differences in age, gender, ALT, AFP, and M2BPGi levels between the two groups at baseline.

[0069] Table 2. Clinical characteristics of serum samples from HCC and control groups (baseline)

[0070] (2-4) Clinical Characteristics of Serum Samples at the Time of HCC Observation Next, we investigated the clinical characteristics of serum samples at the time of HCC onset. The results are shown in Table 3. The mean M2BPGi-Qt value in the HCC group was significantly higher than that in the control group (HCC group: 3.24 ± 3.1, control group: 1.44 ± 0.7, p = 0.001). Additionally, significant differences were observed in ALB and AFP-L3% values ​​between the HCC and control groups.

[0071] Table 3. Clinical characteristics of serum samples from HCC and control groups (at the time of HCC observation)

[0072] On the other hand, although the AFP-L3% value is a well-known HCC marker, the AFP-L3% value was zero in 23 of the 42 HCC patients in this study. This means that in this study, HCC positivity was confirmed relatively early through regular follow-up examinations, and therefore the AFLP-L3% value, which increases depending on the size of solid tumors, was below the lower limit of detection. It also means that, as is well known, AFLP-L3% is not suitable for HCC risk surveys.

[0073] (2-5) Glycosylation profiling of M2BP in serum samples Analysis similar to that in 1 above was performed using serum samples (1 μL) from the HCC group and the control group instead of the enriched M2BP solution. The results are shown in Table 4. In the table, p values ​​of <0.001 are underlined. There were differences in signal intensity between samples, but normalization using the average signal of 45 lectins revealed statistically significant differences in the signals of 17 types of lectins between the HCC group and the control group. For WFA, an M2BP-binding lectin, p = 2.14 × 10 －４ , and there was a significant difference in the measured values ​​of M2BPGi-Qt (Table 3, p = 6.79 × 10 －４ ) were comparable to those in the control group. The asialoglycan-recognizing lectins ECA, BPL, and TJA-II were also significantly elevated in the HCC group. In addition, the fucose-binding lectins PSA, LCA, AOL, and AAL were significantly elevated in the HCC group, while the α2,3 sialic acid-binding lectin ACG and the α2,6 sialic acid-binding lectins SNA and TJA-I were significantly elevated in the control group.

[0074] Table 4. Glycosylation profiling of M2BP in HCC and control groups

[0075] These results suggest that the combined use of multiple lectins may enable more accurate diagnosis of HCC.

[0076] <3. Fully Automated Glycoprofiling of M2BP in Serum Samples> Using a combination of multiple lectins, we performed fully automated glycan profiling using multilectin bead array technology, a format that can be used in clinical settings. In this analysis, we excluded three lectins with overlapping glycan specificities (PSA vs. LCA, TJA-II vs. BPL, and SNA vs. TJA-I) from the 17 lectins that showed significant differences in the results described above (2) and two lectins with high noise levels (DSA and UDA) in the GlycoBIST analysis. A bead array chip, GlycoBIST, containing 12 lectin beads, anti-M2BP antibody beads, biotinylated BSA (positive), and unlabeled BSA (negative) beads was prepared and analyzed using the LuBEA-VIII (Precision System Science) automated analyzer dedicated to BIST. Probe beads for immobilizing lectins and antibodies were prepared by immobilizing the beads with proteins appropriate for each probe and then drying. For multilectin bead array analysis, half of the M2BP immunoprecipitated from 1 μL of serum was diluted to 160 μL with TBSTx and added to 40 μL of probe buffer (1% Triton X-100, 0.5 M glycine, 1 mM CaCl in TBS). ２ , 1 mM MnCl ２The GlycoBIST chip, M2BP sample, primary antibody solution, secondary antibody solution, mixed substrate solution (Bio-Rad Laboratories), and washing buffer (0.1% Triton X-100 in TBS) were placed in the LuBEA-VIII instrument. The reaction time was 10 minutes, and the measurement was automatically processed using a predefined program. The net lectin signals were obtained by subtracting both the BSA signal and the signal for each lectin in PBS (a negative control sample for immunoprecipitation) from the obtained lectin signals. The net lectin signals were normalized by the average of all 12 lectin signals and used in subsequent data analysis. Boxplots of the net lectin signals for each lectin tended to be consistent with the boxplots for the results obtained by the lectin microarray analysis in 2 above (data not shown). Similar analyses were also possible using values ​​normalized by the signal from anti-M2BP antibody beads instead of the average of all lectin signals, but better results were obtained when the average of all lectin signals was used (data not shown).

[0077] Figure 3 shows the results of principal component analysis (PCA) performed using the glycosylation profiles of all 12 M2BPs as the training dataset. The left panel shows plots of serum samples from the HCC and control groups, and the right panel shows plots of the 12 lectins. The PCA biplot separated the HCC group (black) from the control group (white) along the axis of principal component 1 (PC1) (PC1 contribution rate: 75.2%). This result reflects the glycosylation signature of M2BPs associated with HCC (hereinafter referred to as "M2BPgs-HCC"). Therefore, for subsequent statistical characterization, a formula for calculating the M2BPgs-HCC score (F-value) based on PC1 was generated as described previously (Shimazaki, H., et al., Molecules, 2024;29(23):5640). The calculation formula used in this example (when six types of lectins (Table 5) were used) is shown below.

[0078] Formula for calculating M2BPgs-HCC score (F value): F = -0.887 x [TJA-1] + 0.062 x [LCA] + 0.325 x [AAL] + 0.065 x [AOL] + 0.266 x [ECA] + 0.169 x [WFA] + 2.6868 (where [lectin] represents the normalized lectin signal value)

[0079] The scores calculated by the above formula revealed that there was a discrepancy in the values ​​due to measurement error in only one case in the HCC group, and this case was excluded from further analysis.

[0080] For each condition listed in Table 5, accuracy was evaluated using Student's t-test and ROC curve as described in the literature (Kuno, A., et al., Clinical Chemistry, 57(1):48-56). As a result, the score of M2BPgs-HCC when all 12 types of lectins were used [p = 1.32 × 10 －９ , AUC = 0.850, sensitivity = 87.8%, specificity = 72.1%] was the most sensitive, while the conventionally used M2BPGi-Qt score [p = 6.79 × 10 －４ , AUC = 0.733, sensitivity = 54.8%, specificity = 81.4%] and AFP-L3% score [p = 6.26 × 10－５ , AUC = 0.726, sensitivity = 45.2%, specificity = 100%]. These results demonstrate that the usefulness of M2BP for HCC diagnosis is significantly improved by using a combination of multiple lectins. Interestingly, based on the characteristics of the ROC curve, M2BPgs-HCC has diagnostic characteristics different from AFP-L3%, suggesting that the combination of M2BPgs-HCC and AFP-L3 may improve the diagnostic score (data not shown). Therefore, PCA was performed on the dataset of 12 lectins and AFP-L3%, and it was revealed that an even better diagnostic score could be obtained by combining the AFP-L3% score with M2BPgs-HCC, which was obtained as PC1 in the PCA plot (p = 5.42 × 10 －１２ , AUC=0.896, sensitivity=85.4%, specificity=81.4%).

[0081] Next, we investigated the minimum number of lectins required for diagnosing HCC. Using sets of 3 to 6 lectins selected from the 12 lectins mentioned above, we performed the same analysis as above. The signal for each lectin was normalized by the average signal for all lectins in each set.

[0082] The lectin combinations and M2BPgs-HCC scores are shown in Table 5. In the table, selected lectins are indicated with a circle. There was little difference in the scores between the use of six lectins (TJA-I, LCA, AAL, ECA, AOL, and WFA) and the use of 12 lectins. When the number of lectins was further reduced, differences were observed depending on the lectin combination, but all scores were significantly superior to those of AFP-L3% and M2BPGi-Qt.

[0083] Table 5. Lectin combinations and M2BPgs-HCC scores

[0084] Next, the results were examined when combined with the AFP-L3% score. The M2BPgs-HCC + AFP-L3% score was calculated using PC1 + PC2 or PC1 - PC2 in the PCA plot. The results are shown in Table 6. Excellent results were obtained with all lectin combinations.

[0085] Table 6. Lectin combinations and M2BPgs-HCC+AFP-L3% scores

[0086] 4. M2BP Glycosylation Profile in HCC Patients After DAA Treatment In the above examples, differences in M2BPgs-HCC between the HCC group and the control group at the time of HCC observation were confirmed. In this example, serum samples obtained from subjects randomly selected from the aforementioned HCC group before DAA treatment, immediately after treatment, SVR12, SVR24, SVR48, and at the time of HCC observation were analyzed in the same manner as in 3 above. The signals of TJA-1, LCA, AAL, AOL, ECA, and WFA (normalized by the average signal values ​​of the six lectins) were compared.

[0087] Representative results are shown in Figure 4. In most cases, the lectin signal pattern remained consistent from before DAA treatment until the time of HCC observation. This contrasts with the significant decrease in M2BPGi after DAA treatment. Without wishing to be bound by any particular theory, it is speculated that the newly identified M2BP glycosylation isomer M2BPgs in this application may be a HCC growth factor, and that in the HCC group, M2BPgs is present at high levels before DAA treatment and does not decrease after DAA treatment, resulting in the development of HCC.

[0088] These results suggest that the M2BPgs-HCC score may be useful for assessing HCC risk independent of DAA treatment, and that the M2BPgs-HCC score may be useful for identifying compounds that reduce the risk of HCC after achieving SVR.

[0089] <5. Fully automated glycan profiling of M2BP in serum samples (2)> For faster and simpler analysis, the serum used in 3 above was directly subjected to measurement (i.e., without immunoprecipitation). 1 μL of serum was diluted to 160 μL with TBSTx, and 40 μL of probe buffer (TBS, 1% Triton X-100, 0.5 M glycine, 1 mM CaCl ２ , 1 mM MnCl ２ ) and used as the measurement sample. Eleven lectins were used in this measurement: AAL, ECA, LCA, AOL, SNA, NPA, ConA, ACG, BPL, ABA, and WFA. A bead array chip packed with a total of 12 types of beads, including 11 types of lectin beads and anti-M2BP antibody beads, was prepared, and measurements of a total of 85 samples from the HCC and control groups were automatically processed in the same manner as described above in Section 3.

[0090] As a result, compared to the immunoprecipitation test in 3 above, the signal intensity of each lectin varied, and a significant decrease in the signal was observed for lectins such as SNA. This was thought to be due to a competitive reaction between the major glycoproteins contained in serum and M2BP, which was the expected result.

[0091] Figure 5 shows the results of principal component analysis (PCA) performed using the glycosylation profiles of all 12 M2BPs as a training dataset. The left panel shows plots of serum samples from the HCC and control groups, and the right panel shows plots of the 12 lectins. The PCA biplot allowed for separation of the HCC group (black) and the control group (white) along the axis of principal component 1. Although AFP-L3% is a well-known HCC marker and LCA is a lectin used as an indicator of HCC, in this analysis, LCA was selected as the most appropriate lectin to characterize the control group, which was an unexpected result. However, no such changes were observed in AAL and AOL, which are fucose-specific lectins like LCA.

[0092] Using the same procedure as in 3 above, calculation formulas were created for the lectin combinations listed in Table 7, and the M2BPgs-HCC score (F value) was calculated. The results are shown in Table 7. As a result of evaluating the accuracy using Student's t-test and ROC curve, the M2BPgs-HCC score was significantly higher than the AFP-L3% score [p=6.26×10 －５ In particular, good results were obtained with the combination of lectins including LCA.

[0093] Table 7. Lectin combinations and M2BPgs-HCC scores

[0094] 6. M2BP Glycosylation Profile in the HCC Group at SVR12 After DAA Treatment The above tests demonstrated that the M2BPgs-HCC score outperforms existing diagnostic markers in distinguishing between HCC and control groups during HCC observation. Furthermore, it was also shown that there was almost no change in the lectin signal pattern from before DAA treatment to the time of HCC observation. However, its applicability to predicting the onset of HCC is unclear. Therefore, a similar analysis to that described in 5 above was performed on a total of 145 cases, including 30 HCC cases and 30 control cases for which serum at SVR12 was available. It was confirmed that the M2BPgs-HCC score also exhibits superior performance in analyses including samples at SVR12. Comparison of score performance for 136 cases for which AFP-L3% values ​​were available revealed that the M2BPgs-HCC score obtained by this method significantly outperformed the M2BPGi-Qt score [p=2.06×10 －２ ], AFP score [p = 3.01 × 10 －１ ], and AFP-L3% score [ p = 7.82 × 10 －５ The performance was confirmed to be far superior to that of the combination of two lectins (LCA and AOL, or LCA and WFA), or the combination of anti-M2BP antibody and AOL (Table 8).

[0095] Table 8. Lectin / anti-M2BP antibody combinations and M2BPgs-HCC scores

[0096] For further application, we also performed an evaluation using only SVR12 samples. As a result, no significant differences were observed in the M2BPGi-Qt score [p=0.658], AFP score [p=0.249], or AFP-L3% score [p=0.3930]. However, significant differences were observed in all M2BPgs-HCC scores obtained using this method, with some lectin sets showing particularly significant differences (Table 9, "*" indicates p<0.001). These results suggest the possibility of predicting the onset of HCC at SVR12.

[0097] Table 9. Lectin / anti-M2BP antibody combinations and M2BPgs-HCC scores

[0098] 7. Analysis Using a Single Lectin Bead Chip For the combination of LCA, AOL, and WFA that provided particularly excellent results in section 6 above, three types of chips were prepared, each loaded with a single lectin bead. These chips were then compared with a multilectin bead array chip loaded with 11 types of lectin beads and anti-M2BP antibody beads, a total of 12 types of beads, to confirm whether any discrepancies in the data existed. Serum samples from 10 randomly selected cases each from the HCC group and the control group (20 cases in total) were analyzed using the multilectin bead array chip, and the LCA, AOL, and WFA measurements were normalized by the average of all the LCA, AOL, and WFA measurements. The same samples were analyzed using an LCA bead-loaded chip, an AOL bead-loaded chip, and a WFA bead-loaded chip, and the measurements from each chip were normalized by the average of the measurements from the three chips.

[0099] The analytical results using the multilectin bead array chip are shown in Figure 6, and the analytical results using the single-lectin bead chip are shown in Figure 7. At first glance, both graphs are consistent, confirming that the same results are obtained when multiple lectins are reacted in parallel and when each lectin is reacted independently. Therefore, when analyzing using a minimum number of lectins, it is possible to obtain data for each lectin using a single-lectin measurement system and combine these data to determine the risk of developing HCC.

Claims

1. A method for determining the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a virological response, comprising: (1) contacting a Mac-2 binding protein in a sample from the subject with at least two lectins, wherein the lectins are selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and (2) measuring the binding of each of the at least two lectins to the Mac-2 binding protein.

2. The method of claim 1, wherein the at least two types of lectins are selected from the following (i) to (vi): (i) TJA-I or SNA, (ii) LCA or PSA, (iii) AAL, (iv) ECA, (v) AOL, and (vi) WFA.

3. The method of claim 1 or 2, wherein the at least two types of lectins include AOL and / or WFA.

4. The method of claim 1 or 2, wherein the at least two lectins include LCA.

5. (3) A method according to any one of claims 1 to 4, further comprising a step of normalizing the measurement values ​​for each of the at least two types of lectins obtained in step (2) by the average of the measurement values ​​for all of the at least two types of lectins.

6. The method according to any one of claims 1 to 5, wherein the sample is blood.

7. The method according to any one of claims 1 to 6, further comprising a step of capturing Mac-2 binding proteins in a sample derived from the subject prior to step (1).

8. A method for screening for a compound that reduces the risk of developing hepatocellular carcinoma in a subject infected with hepatitis C virus who has achieved a virological response, the method comprising the steps of: (1) contacting a candidate compound with hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts derived from hepatocellular carcinoma tissue; (2) capturing a Mac-2 binding protein derived from the hepatocytes, hepatic stellate cells, and / or hepatic fibroblasts; (3) contacting the Mac-2 binding protein with at least two types of lectin, wherein the lectin is selected from the group consisting of TJA-I, AAL, ECA, LCA, AOL, PSA, SNA, GSL-II, NPA, ConA, ACG, BPL, TJA-II, ABA, UDA, DSA, and WFA; and (4) measuring binding of each of the at least two types of lectin to the Mac-2 binding protein.

9. The method of claim 8, wherein the at least two types of lectins are selected from the following (i) to (vi): (i) TJA-I or SNA, (ii) LCA or PSA, (iii) AAL, (iv) ECA, (v) AOL, and (vi) WFA.

10. The method of claim 8 or 9, wherein the at least two types of lectins include AOL and / or WFA.

11. A method according to any one of claims 8 to 10, further comprising the step of (5) normalizing the measurement values ​​for each of the at least two types of lectins obtained in step (4) by the average of the measurement values ​​for all of the at least two types of lectins.

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