AU2018229466A1 - Method for determining tissue regeneration state of living body organs - Google Patents

Abstract

Disclosed is a method for determining a tissue regeneration state, including the steps of measuring opioid growth factor receptor-like 1 (OGFRL1) levels in a body fluid collected from a subject, comparing the obtained OGFRL1 values with a reference value, and indicating that tissue regeneration is accelerated in the subject when the OGFRL1 value is equal to or greater than the reference value.

Description

The present disclosure relates to a method for determining a tissue regeneration state. The present disclosure relates to a method including a step of detecting opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject.

BACKGROUND [0002]

When a tissue is injured or lost, remaining cells of the injured or defective tissue proliferate, resulting in regeneration and repair. When this response is insufficient, regeneration/repair treatment of injured or defective tissue is attempted by transplantation and the subsequent proliferation of cells derived from embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells). The degree of cell proliferation in the injured or defective tissue reflects a regeneration state of the tissue and can be used as an important marker for determining therapeutic effects and predicting prognosis of patients.

[0003]

An example of a marker for evaluating tissue regeneration is α-fetoprotein (AFP), which production has been reported to increase in a recovery stage from acute liver injury induced by D-galactosamine administration into rats (Noguchi T. “a-Fetoprotein producing cells during liver restitution after acute D-galactosamine hepatitis in the rat immunoperoxidase and autoradiographic studies Journal of Japanese Society of Gastroenterology 75: 1972-1982, 1978,). Clinically, it is also known that the serum AFP

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2018229466 12 Sep 2018 levels increase in a recovery stage from acute severe liver injury (Kakisaka K, et al. “Alpha-fetoprotein: a biomarker for the recruitment of progenitor cells in the liver in patients with acute liver injury or failure.” Hepatology Research 45: E12-E20, 2015) and cirrhosis that is a terminal stage of chronic liver injury (Liu YR, et al. “Alpha-fetoprotein level as a biomarker of liver fibrosis status: a cross-sectional study of 619 consecutive patients with chronic hepatitis B.” BMC Gastroenterol 14: 145, 2014).

Regarding OGFRL1, it has been reported that its expression is up-regulated in a cancer stem cell fraction (WO 2013/122140 A and JP 2015-107918 A).

SUMMARY [0006]

Since the expression of AFP frequently and remarkably increases in liver cancer cells as described above, it is difficult to determine whether the increase in serum AFP levels represents production by liver cancer cells, or reflects recovery from severe liver injury . Therefore, AFP is not suitable as a marker for determining proliferation of non-tumor cells, and is currently used exclusively as a tumor marker. As described above, there is no specific marker for evaluating the regeneration or repair status of the liver that has been suffered from acute or chronic injury, and it is difficult to determine therapeutic effects and predict prognosis of patients with severe liver injury. Therefore, development of a useful market that can distinguish tissue regeneration from tumor cell proliferation has been urgently desired.

[0007]

Tissue biopsy has been used to determine cell proliferation status in the regenerating tissue collecting from a patient's body. However, tissue biopsy has problems of being highly invasive and painful, and sometimes involving severe complications such

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2018229466 12 Sep 2018 as massive bleeding. Therefore, a method for evaluating cell proliferation with less invasiveness is eagerly desired.

[0008]

From these points of view, it is the object of the present disclosure to provide a tissue regeneration marker reflecting cell proliferation status. It is also the object of the present disclosure to provide a tissue regeneration marker which is not affected by tumor cell proliferation and can be evaluated with low invasiveness.

[0009]

The first embodiment of the present disclosure relates to a method for determining a tissue regeneration state. The method comprises the steps of: (1) measuring opioid growth factor receptor-like 1 (OGFRL1) levels in a body fluid collected from a subject,(2) comparing the obtained OGFRL1 values with a reference value, and (3) indicating that tissue regeneration is accelerated in the subject when the OGFRL1 value is equal to or greater than the reference value.

[0010]

The second embodiment of the present disclosure relates to a method including the step of detecting opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject, wherein an enhancement of tissue regeneration in the subject is indicated by the detection of OGFRL1.

According to this embodiment, it is possible to suggest accelerated tissue regeneration, by detecting OGFRL1 in a body fluid.

[0011]

According to the present disclosure, it is possible to provide a tissue regeneration marker.

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BRIEF DESCRIPTION OF THE DRAWINGS [0012]

Fig. 1A shows a protein blot image of OGFRL1 present in the serum from mice injected with carbon tetrachloride or diethylnitrosamine (DEN).

Fig. IB shows a histogram of luminescence intensities representing relative expression levels of OGFRL1 shown in each lane of Fig. 1A. “Minus (-)” in the figure indicates control mice without carbon tetrachloride administration, or mice to which DEN was administered but liver cancer has not developed. “Plus (+)” indicates mice administered with carbon tetrachloride or mice in which liver cancer has developed by DEN administration.

Fig. 2A shows a protein blot image of OGFRL1 present in serum exosomes or liver tissues from carbon tetrachloride-administered mice. Fig. 2B shows a histogram of luminescence intensities representing relative expression levels of OGFRL1 shown in each lane of Fig. 2A. “Minus (-)” and “plus (+)” in the figure indicate mice without and with carbon tetrachloride administration, respectively.

Fig. 3A shows a protein blot image of OGFRL1 present in serum exosomes obtained from mice administered with different concentrations of carbon tetrachloride. Fig. 3B shows a histogram of luminescence intensities representing relative expression levels of OGFRL1 shown in each lane of Fig. 3A. The numbers in the figure (1 to 13) indicate the individual numbers of each mouse.

Fig. 4 shows the luminescence intensities measured by a sandwich enzyme-linked immunosorbent assay (ELISA) method of serum OGFRL1 using anti-OGFRLl (344-362) antibodies and anti-CD9 antibodies. “Minus (-)” and “plus (+) in the figure indicate mice without and with carbon tetrachloride administration, respectively. The numbers in the figure (1 to 5) indicate the individual numbers of each mouse.

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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0013]

1. First Embodiment

The first embodiment of the present disclosure includes a step of measuring opioid growth factor receptor-like 1 (OGFRL1) levels in a body fluid collected from a subject. [0014]

In the case of human, examples of OGFRL1 include a protein registered in GenBank under accession number Q5TC84.1 provided by The National Center for Biotechnology Information, and its isoforms. In the case of mouse, examples include a protein registered in GenBank with accession number Q8VE52.2, and its isoforms. In the present disclosure, OGFRL1 includes variants of proteins registered with the above accession numbers and orthologs.

[0015]

The subject is an individual whose state of tissue regeneration is evaluated. The individual is not particularly limited, and examples thereof include mammals such as human, monkey, mouse, rat, dog, cat, rabbit, bovine, horse, goat, sheep and pig, birds such as chicken, and the like. The individual is preferably a mammal such as a human, a monkey, a mouse, a rat, a dog, a cat, a bovine, a horse or a pig, more preferably a human, a monkey, a mouse, a rat, a dog, a cat or the like, further preferably a human, a monkey, a mouse or a rat, and most preferably a human. The age and gender (male or female) of the individual do not matter.

[0016]

The subject may include both an individual having a disease and an individual having no disease. The subject is preferably an individual who does not have a tumor,

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2018229466 12 Sep 2018 more preferably does not have a malignant tumor, and further preferably does not have a malignant tumor in the liver. It is preferable that the subject has a non-neoplastic disease. The non-neoplastic disease is not limited as long as it is a disease other than tumor. For example, the non-neoplastic disease is a disease exhibiting hepatic dysfunction, and examples of the types of hepatic dysfunction include acute hepatitis, chronic hepatitis, cirrhosis, fatty liver disease, and the like. Examples of the causes of hepatic dysfunction include viral, alcoholic, drug-induced, autoimmune, disorder of glucose metabolism, disorder of lipid metabolism, disorder of amino acid metabolism, metal metabolism disorder, cholestasis, impaired hepatic blood flow, and the like.

[0017]

The body fluid can be collected from the subject and is not limited as long as it can detect OGFRL1. Examples of the body fluid include blood samples, ascites, pleural effusion, pericardial effusion, cerebrospinal fluid, interstitial fluid, lymph fluid, urine, and the like. The blood samples include whole blood, serum, and plasma as well. The body fluid is more preferably, serum or plasma.

[0018]

It is preferable that the body fluid contains extracellular vesicles. The extracellular vesicles are also called extracellular secretory vesicles and include exosomes, microvesicles, apoptotic bodies, and the like. Extracellular vesicles can be recovered, for example, using a commercially available exosome precipitation reagent such as ExoQuick (System Biosciences).

[0019]

The tissue is not limited as long as it exists in the body of the subject. Preferably, the tissue is in an organ in which the non-neoplastic disease is present. More preferably, the organ is the liver.

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2018229466 12 Sep 2018 [0020]

Tissue regeneration refers to a process in which an injured or defective tissue returns to its original state quantitatively and functionally, or a state where it returns to its original. In order to return to its original state both quantitatively and functionally, it is desirable that the remaining cells constituting the tissue or the transplanted cells proliferate to restore the size of the organ, and also exhibit the function specific to the tissue to restore the impaired function.

[0021]

The description that tissue regeneration is accelerated indicates that the remaining cells constituting the tissue or the transplanted cells proliferate actively in the injured or defective tissue to return to its original state both quantitatively and functionally, and means that the repair of the tissue is proceeding smoothly, that is, the subject is in a recovery stage from tissue disorder.

[0022]

The measurement method of OGFRL1 is not limited as long as the measured value of OGFRL1 can be obtained. Here, the measured value of OGFRL1 is a value that reflects the amount or concentration of OGFRL1. For example, in order to obtain a measured value of OGFRL1, antibodies capable of specifically binding to OGFRL1 protein, that is, anti-OGFRLl antibodies can be used. Examples of the method for obtaining the measured value of OGFRL1 protein by the anti-OGFRLl antibodies include an ELISA and a protein blot analysis.

[0023]

For example, in the case of obtaining the measured value of OGFRL1 by the ELISA method, the order of mixing a body fluid or extracellular vesicles recovered from the body fluid and the anti-OGFRLl antibodies is not particularly limited, and these may

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2018229466 12 Sep 2018 be mixed substantially at the same time, or may be mixed successively. Hereinafter, the present embodiment will be described with a method of detecting OGFRL1 protein in a body fluid as an example.

[0024]

In this embodiment, a complex of the OGFRL1 protein in the body fluid and the anti-OGFRLl antibodies is first formed and then the complex is immobilized on a solid phase, or the anti-OGFRLl antibodies are immobilized on a solid phase in advance, and the OGFRL1 protein in the body fluid can be coupled with the immobilized anti-OGFRLl antibodies. More preferred is an embodiment in which the complex is first formed and then the complex is immobilized on a solid phase. Then, the complex immortalized on a solid phase or a complex formed on a solid phase is detected by a method known in the art, whereby the measured value of OGFRL1 protein contained in the body fluid can be obtained.

[0025]

In the case where a complex of the OGFRL1 protein in the body fluid and the anti-OGFRLl antibodies is first formed and then the complex is immobilized on a solid phase, anti-OGFRLl antibodies labeled with biotin or the like can be used to make a contact with the OGFRL1 protein in the body fluid to form a complex. By separately binding avidin or streptavidin (hereinafter also referred to as “avidins”) to the solid phase in advance, the complex can be immobilized on the solid phase via binding between biotin and avidins.

[0026]

In the case where the anti-OGFRLl antibodies are immobilized on the solid phase in advance, the method of immobilization of the anti-OGFRLl antibodies to the solid phase is not particularly limited. For example, the anti-OGFRLl antibodies may be

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2018229466 12 Sep 2018 directly bound to the solid phase, or the anti-OGFRLl antibodies and the solid phase may be indirectly bonded with another substance interposed therebetween. Examples of the direct binding include physical adsorption and the like. Examples of the indirect binding include a bond via coupling with avidins. In this case, by labeling the anti-OGFRLl antibodies with biotin in advance and separately binding avidins to the solid phase, the anti-OGFRLl antibodies and the solid phase can be indirectly bound via the bond between the biotin and the avidins. In this embodiment, it is preferable that the bond between the anti-OGFRLl antibodies and the solid phase is an indirect binding between biotin and avidins.

[0027]

The material of the solid phase is not particularly limited, and it can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, and the like. Examples of the organic polymer compound include latex, polystyrene, polypropylene, and the like. Examples of the inorganic compound include magnetic bodies (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include insoluble agarose, insoluble dextran, gelatin, cellulose, and the like. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include particles, membranes, microplates, microtubes, test tubes, and the like. Among them, particles are preferable, and magnetic particles are particularly preferable.

[0028]

In this embodiment, bound/free (B/F) separation for removing unreacted free components that do not form a complex may be carried out after the formation of the complex, preferably after formation of the complex and before detection of the labeling substance. The unreacted free component refers to a component that does not constitute a

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2018229466 12 Sep 2018 complex. Examples of the unreacted free component include an anti-OGFRLl antibodies not bonded to the OGFRL1 protein, and the like. The means of B/F separation is not particularly limited, and when the solid phase is a particle, B/F separation can be performed by recovering only the solid phase capturing the complex by centrifugation. When the solid phase is a container such as a microplate or a microtube, B/F separation can be performed by removing a liquid containing an unreacted free component. When the solid phase is a magnetic particle, B/F separation can be performed by aspirating and removing a liquid containing an unreacted free component using a nozzle while magnetically constraining the magnetic particles with a magnet. This method is preferable from the viewpoint of automation. After removing the unreacted free component, the solid phase capturing the complex may be washed with a suitable aqueous medium such as phosphate buffered saline (PBS).

[0029]

In this embodiment, the complex can be detected, for example, using an anti-OGFRLl antibodies coupled with a labeling substance, or using labeled anti-immunoglobulin antibodies capable of binding to the unlabeled anti-OGFRLl antibodies or the like, but it is preferable to use labeled anti-OGFRLl antibodies. An epitope in the OGFRL1 protein detected by the labeled anti-OGFRLl antibodies is preferably different from an epitope in the OGFRL1 protein of the secondary anti-OGFRLl antibodies that bind to the solid phase.

[0030]

The labeling substance used for the label of anti-OGFRLl antibodies or the anti-immunoglobulin antibodies is not particularly limited as long as the labeling substance generates a detectable signal. For example, it may be a substance which itself generates a signal (hereinafter also referred to as “signal generating substance”) or a substance which

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2018229466 12 Sep 2018 catalyzes the reaction of other substances to generate a signal. Examples of the signal generating substance include fluorescent substances, radioactive isotopes, and the like. Examples of the substance that catalyzes the reaction of other substances to generate a detectable signal include enzymes. Examples of the enzymes include alkaline phosphatase (ALP), peroxidase, β-galactosidase, luciferase, and the like. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), fluorescent proteins such as enhanced green fluorescent protein (EGFP), and the like. Examples of the radioisotopes include

I, C, P, and the like. Among them, an enzyme is preferable as a labeling substance, and ALP is particularly preferable.

[0031]

The labeled anti-OGFRLl antibodies are obtained by labeling the anti-OGFRLl antibodies with the above-mentioned labeling substance by a labeling method known in the field of immunoassay technology. Labeling may also be performed using a commercially available labeling kit or the like. As the labeled immunoglobulin antibodies, the same method as the labeling of the anti-OGFRLl antibodies may be used, or a commercially available product may be used.

[0032]

In this embodiment, the value of OGFRL1 contained in a body fluid can be measured by detecting a signal generated by the labeling substance bound to the anti-OGFRLl antibodies contained in the complex,. The phrase “detecting a signal” herein includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the intensity of the signal. Semi-quantitative detection means to show the intensity of the signal in stages such as “no signal generated”, “weak”, “medium”, “strong”, and the like.

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2018229466 12 Sep 2018 [0033]

Methods for detecting a signal themselves are known in the field of immunoassay technology. In this embodiment, a measurement method according to the type of signal derived from the labeling substance may be appropriately selected. For example, when the labeling substance is an enzyme, signals such as light and color generated by reacting with a substrate for the enzyme can be measured by using a known apparatus such as a luminometer or a spectrophotometer.

[0034]

The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when ALP is used as the enzyme, examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (disodium

4-chloro-3 -(methoxyspiro [ 1,2-dioxetane-3,2 ’ -(5 ’ -chloro)tricyclo [3.3.1.13,7] decan] -4-yl)ph enyl phosphate) and CSPD (registered trademark) (disodium

-(4-methoxyspiro [ 1,2-dioxetane-3,2-(5 ’ -chloro)tricyclo [3.3.1.13,7] decan] -4-yl)phenyl phosphate), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate and p-nitrophenyl phosphate. Particularly preferred is CDP-Star (registered trademark). The luminescence of the substrate is preferably detected with a luminometer.

[0035]

When the labeling substance is a radioactive isotope, radiation as a signal can be measured using a known apparatus such as a scintillation counter. When the labeling substance is a fluorescent substance, fluorescence as a signal can be measured using a known apparatus such as a fluorescence microplate reader. The excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of

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2018229466 12 Sep 2018 fluorescent substance used.

[0036]

The detection result of the signal can be used as the measured value of OGFRL1 protein. For example, when quantitatively detecting the intensity of a signal, the measured value itself of the signal intensity or the value calculated from the measured value of the signal intensity can be used as the measured value of OGFRL1 protein. Examples of the value calculated from the measured value of the signal intensity include a value obtained by subtracting the measured value of the signal intensity of the negative control sample from the measured value of the signal intensity of the test sample, a value obtained by dividing the measured value of the signal intensity of the test sample by the measured value of the signal intensity of the positive control sample, combinations thereof, and the like. Examples of the negative control sample include body fluids obtained from healthy individuals and the like. Examples of the positive control sample include a body fluid containing OGFRL1 protein at a predetermined concentration.

[0037]

The measured value of OGFRL1 protein in the body fluid can be calculated by preparing a calibration curve from measured values of the signal intensity of a positive control containing known concentrations of OGFRL1 protein and applying the measured value of the intensity of the signal of OGFRL1 protein in the test sample to the calibration curve. The measured value of OGFRL1 protein in the body fluid can be calculated by obtaining a regression equation from measured values of the signal intensity of a positive control containing known concentrations of OGFRL1 protein without preparing a calibration curve, and applying the measured value of the intensity of the signal of OGFRL1 protein in the test sample to the regression equation. When the measured value is represented by concentration, it may be a molar concentration or a ratio (mass/volume)

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2018229466 12 Sep 2018 of mass per constant volume of a biological sample.

[0038]

The anti-OGFRLl antibodies are not limited as long as they specifically bind to the OGFRL1 protein, and polyclonal antibodies, a monoclonal antibody, and a fragment thereof (for example, Fab, F(ab)2, or the like) obtained by immunizing non-human animals using an OGFRL1 protein or a part thereof as an antigen can be used. Also, immunoglobulin classes and subclasses are not particularly limited. It may also be a chimeric antibody. Further, it may also be scFv or the like.

[0039]

Examples of the antigen used for preparing anti-OGFRLl antibodies include OGFRL1 proteins registered in GenBank with accession number Q5TC84.1 for human and with accession number Q8VE52.2 for mouse, respectively. For example, a peptide consisting of 19 amino acids from the 344th to 362nd positions on the amino acid sequence of mouse OGFRL1 represented by the accession number Q8VE52.2 can be used as the antigen. The peptide can be synthesized by a known method.

[0040]

In this embodiment, either antibodies binding to OGFRL1 in the body fluid or labeled anti-OGFRLl antibodies may be replaced with antibodies that bind to a protein present in extracellular vesicles or the like. Examples of the protein present in extracellular vesicles include CD9 and the like.

This embodiment further includes the step of comparing the measured value with a reference value.

[0041]

The reference value can be preset. For example, a group of body fluid collected from individuals in whom regeneration is accelerated in a tissue other than tumor is set as a

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2018229466 12 Sep 2018 positive control group, and another group of body fluid collected from healthy individuals is set as a negative control group. Then, a value that can most accurately classify the positive control group and the negative control group can be adopted as a reference value. Here, “the value that can most accurately classify” can be appropriately set based on indices such as sensitivity, specificity, positive predictive value, negative predictive value, and the like, depending on the purpose of the examination. Further, the reference value can be determined by a ROC (receiver operating characteristic) curve, a discriminant analysis method, a mode method, the Kittier method, a 3σ method, a p-tile method, and the like. The reference value is preferably set for each body fluid.

[0042]

In this embodiment, when the measured value is equal to or greater than the reference value, it is indicated that tissue regeneration is accelerated in the subject. Herein, indicating that tissue regeneration is accelerated in the subject includes determining that tissue regeneration is accelerated in the subject.

[0043]

Furthermore, when it is indicated that tissue regeneration is accelerated in the subject, this embodiment may indicate that the subject is in a recovery stage from disorder of the tissue. Herein, indicating that the subject is in a recovery stage from disorder of the tissue includes determining that the subject is in a recovery stage from disorder of the tissue.

2. Second Embodiment [0044]

The second embodiment of the present disclosure relates to a method including a step of detecting opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject. By the method described in the first embodiment above, it is possible to

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2018229466 12 Sep 2018 detect OGFRL1 in the body fluid. Here, the detection includes whether or not OGFRL1 is present in the body fluid is determined qualitatively, by the method described in the first embodiment, in addition to the method for obtaining the measured value of OGFRL1 in the body fluid described in the first embodiment.

[0045]

In this embodiment, when OGFRL1 is detected in the body fluid, accelerated tissue regeneration is indicated in the subject. Herein, indicating that tissue regeneration is accelerated in the subject includes determining that tissue regeneration is accelerated in the subject.

[0046]

Furthermore, when it is indicated that tissue regeneration is accelerated in the subject, this embodiment may indicate that the subject is in a recovery stage from disorder of the tissue. Herein, indicating that the subject is in a recovery stage from disorder of the tissue includes determining that the subject is in a recovery stage from disorder of the tissue.

For the terms used in the second embodiment, the description in the first embodiment is incorporated.

EXAMPLES [0047]

Hereinafter, the present disclosure will be described in more detail by way of examples, but the present disclosure is not to be construed as being limited to the examples.

[0048]

1. Example 1

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In order to verify that OGFRL1 can be used as a marker to determine cell proliferation status other than tumor, detection of OGFRL1 in serum was carried out using mice with liver injury induced by carbon tetrachloride administration and mice injected with DEN, a hepatic carcinogen.

[0049]

-1. Preparation of Anti-OGFRL 1 Antibodies

A peptide consisting of 19 amino acids from the 344th to 362nd positions among mouse OGFRL1 protein consisting of full length 464 amino acids was synthesized, a rabbit was immunized with this synthetic peptide, and the antibody titer of the serum obtained after 84 days was confirmed by ELISA. Using the synthetic peptide used as an immunogen, the rabbit serum was purified by passing through a column.

[0050]

1-2. Preparation of Mice with Carbon Tetrachloride-Induced Liver Injury

Carbon tetrachloride was diluted by mixing with a 3-times liquid volume of olive oil to prepare a 25% carbon tetrachloride solution. Using the prepared carbon tetrachloride solution, 1 mL of carbon tetrachloride per kg body weight was subcutaneously injected to male C57BL/6J mice (CLEA Japan, Inc.) weighing 20 to 25 g under isoflurane inhalation anesthesia. Liver tissues were excised under isoflurane inhalation anesthesia from control mice without carbon tetrachloride intoxication and mice with carbon tetrachloride-induced liver injury at 24 hours after carbon tetrachloride administration (3 mice each).

[0051]

-3. Induction of Liver Cancer by Administration of DEN

Male C57BL/6J mice at 3 weeks old were administered intraperitoneally withlO pg DEN per kg body weight as a single dose. Ten months after administration of DEN,

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2018229466 12 Sep 2018 the abdomens were opened under isoflurane anesthesia, and the presence of tumor on the liver surface was examined macroscopically. The presence or absence of liver cancer was histologically confirmed for the excised liver, and the DEN-administered mice were divided into two groups depending on the presence or absence of liver cancer.

[0052]

1- 4. Collection of Serum

To collect serum, the abdomens of the mice were opened under isoflurane anesthesia and blood was collected from the inferior vena cava. The collected blood was allowed to stand at room temperature for 30 minutes or more, and then serum and clot were separated by centrifugation at 1,200 x g for 20 minutes. The obtained serum was stored at -20°C until use.

[0053]

-5. Protein Blot Analysis

A homogenization buffer (final concentration: 20 mM Tris-HCl pH 7.5, 60 mM beta-glycerophosphate, 10 mM ethylene glycol tetraacetic acid, 10 mM magnesium chloride, 10 mM sodium fluoride, 2 mM dithiothreitol, 1.0% NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 x proteinase inhibitor cocktail [aprotinin, leupeptin, and pepstatin A]) was added to the serum and mixed, then the mixture was centrifuged at 4°C, 13,500 rpm for 10 minutes. An equal amount of 2 x Laemmli sample buffer (final concentration: 32.9 mM Tris-HCl pH 6.8, 1.05% SDS, 2.5%

2- mercaptoethanol, 13.15% glycerol, 0.005% bromophenol blue) was added to the obtained supernatant and mixed, then the mixture was heated at 98°C for 5 minutes. Samples corresponding to 50 pg of protein were subjected to a 10% polyacrylamide/SDS gel electrophoresis, and then transferred to a PVDF membrane. The membrane was shaken in blocking buffer (3% bovine serum albumin [BSA]/PBS) at room temperature for

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2018229466 12 Sep 2018 hour, anti-OGFRLl (344-362) antibodies diluted 3,000-fold were added, and allowed to react at 4°C for 16 hours. After washing the membrane, a peroxidase-labeled secondary antibodies diluted 10,000-fold were added and allowed to react at room temperature for 45 minutes. The membrane was washed and then reacted with Immobilon Western Chemiluminescent HRP Reagent (Merck-Millipore) to detect a band indicating OGFRL1 protein.

[0054]

1- 6. Results

Results of protein blot analysis of sera obtained from non-injected and carbon tetrachloride-injected (3 mice each), and those from mice without or with DEN-induced liver cancer (3 mice each) are shown in Fig. 1. As is apparent from Fig. 1, OGFRL1 was not detected in the sera from mice without carbon tetrachloride intoxication, but the amounts of OGFRL1 in the sera increased by administration of carbon tetrachloride. In contrast, OGFRL1 was not detected in the sera from mice with DEN-induced liver cancer. These results indicate that OGFRL1 detected in the serum can be used as a marker for evaluating cell (hepatocyte) proliferation status other than tumor.

[0055]

2. Example 2

Detection of OGFRL1 in liver tissue and serum exosomes was performed in carbon tetrachloride-administered mice.

[0056]

2- 1. Extraction of Proteins from Liver Tissue

The excised liver tissue was homogenized in the homogenization buffer, 2 x Laemmli sample buffer was added thereto, and the mixture was heat-treated as described in Example 1.

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2-2. Collection of Serum Exosomes and Detection of OGFRL1

Carbon tetrachloride-administered mice were prepared according to the method described in Example 1. Exosomes were recovered from the sera of the mice using an exosome precipitation reagent (ExoQuick [System Biosciences]) according to the attached protocol. Specifically, after removing living cells or cell fragments from serum samples by centrifugation, the supernatant was mixed with ExoQuick reagent and allowed to react at 4°C for 30 minutes. The reactant was centrifuged at 1,500 x g for 30 minutes to obtain a precipitate containing exosome. To the precipitate was added the homogenization buffer described in Example 1 to dissolve the precipitate, an equal amount of 2 x Laemmli sample buffer was added, and then the mixture was heated at 98°C for 5 minutes.

[0058]

2-3. Protein Blot Analysis

Protein blot analysis was performed using the method described in Example 1. [0059]

2-4. Results

The results are shown in Fig. 2. As is apparent from Fig. 2, OGFRL1 markedly increased in the serum exosomes at 24 hours after carbon tetrachloride administration. When the amounts of OGFRL1 protein in the liver tissue per unit liver weight were compared, it was rather decreased in the liver tissue at 24 hours after administration of carbon tetrachloride as compared to in the liver tissue without carbon tetrachloride administration. These results indicate that the increase in the concentration of OGFRL1 in the serum or serum exosomes, but not the OGFRL1 content in the liver tissue, reflects the proliferation state of cells (hepatocytes).

[0060]

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3. Example 3

Different amounts of carbon tetrachloride were administered to mice, and the amounts of OGFRL1 in serum exosomes from each mouse were compared.

[0061]

3-1. Carbon Tetrachloride Administration to Mice and Detection of OGFRL1 in Serum Exosomes

A carbon tetrachloride solution was prepared in the same manner as in Example 1. In addition to a group to which 1 mL of carbon tetrachloride per kg body weight was administered to induce a moderate degree of liver injury, additional groups of mice were set to which either 0.5 mL/kg body weight as a half dose or 2 mL/kg body weight of carbon tetrachloride as a double dose was administered. The OGFRL1 protein in serum exosomes from each group of mice at 24 hours after administration was detected by protein blot analysis using the same method as described in Example 1 and Example 2.

[0062]

Here, according to the results of a previous study using other mice, it is known that all the mice can survive and recover when administered with 0.5 or 1 mL/kg body weight of carbon tetrachloride, whereas the majority of mice fall into severe liver injury and die by administration of 2 mL/kg body weight of carbon tetrachloride. That is, in the case of administration of 2 mL/kg body weight of carbon tetrachloride, sufficient tissue regeneration does not occur, and the mice cannot recover from liver injury.

[0063]

3-2. Results

The results are shown in Fig. 3. As shown in Fig. 3, the amounts of OGFRL1 protein in the serum exosomes significantly increased in the mice with 0.5 mL or 1.0 mL/kg body weight of carbon tetrachloride administration, whereas the amounts of

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OGFRL1 protein in the serum exosomes markedly decreased in the mice administered with 2.0 mL/kg body weight of carbon tetrachloride. That is, it was clearly indicated that the amounts of OGFRL1 protein in the serum increase in mice that can recover from liver injury due to cell (hepatocyte) proliferation, and conversely, the concentration of OGFRL1 in the serum decrease in mice that cannot recover from liver injury. This indicates that evaluation of therapeutic effect and prediction of prognosis in subjects with liver injury can be made by measuring the concentration of OGFRL1 in serum exosomes.

[0064]

4. Example 4

Serum OGFRL1 in non-injected (2 mice) or carbon tetrachloride-injected mice (3 mice) was detected using a sandwich ELISA method. Anti-OGFRLl (344-362) antibodies were used as the capture antibody, and anti-CD9 antibodies (BioLegend #312102) were used as the detection antibody to react with CD9 that is known to be present in the exosome.

[0065]

4-1. ALP Labeling of Detection Antibody

Alkaline Phosphatase Labeling Kit-NH2 (Dojindo kit #LK12) was used to label the detection antibody with ALP. The labeling method was in accordance with the protocol attached to the kit. The labeled antibodies were stored at 4°C after adding the storage buffer attached to the kit.

[0066]

4-2. Purification of Capture Antibody

Five hundred pL of PBS was added to Amicon ultra 50 kDa column, and centrifuged at 14000 x g for 5 minutes. The same volume of PBS was added again, and the above centrifugation was repeated to wash the column five times in total. The column

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2018229466 12 Sep 2018 was inverted and centrifuged at 1000 x g for 10 minutes. Anti-OGFRLl (344-362) antibodies (22.4 pg) as a capture antibody were permeated through the column, and 492 pL of carbonate buffer was added. After centrifugation at 14000 x g for 5 minutes, 400 pL of carbonate buffer was added and centrifuged again. This operation was repeated twice. The column was inverted and centrifuged at 1000 x g for 10 minutes to recover the purified antibodies. The protein concentration of the purified antibodies was determined by measuring the absorbance using Nanodrop (Thermo Fisher Scientific). The results were as follows.

Concentration: 0.5154 pg/pL (average of five measurements)

Yield: 33 pL

Total protein content: 17.0082 pg [0067]

4-3. Immobilization of Capture Antibody to Solid Phase

The concentration pf anti-OGFRLl (344-362) antibodies as a capture antibody were adjusted to 5 pg/mL with carbonate buffer and added to each well of the ELISA plate to be 90 pL/well. After standing at 4°C for 15 hours, the wells were washed three times with 300 pL/well of PBS containing 0.05% (w/v) Tween 20 (PBS-T).

[0068]

4-4. ELISA Measurement

PBS-T containing 1% BSA was added to the capture antibody solid phase plate prepared in 4-3. to be 200 pL/well. After blocking at room temperature for 1.5 hours, the plate was washed three times with 300 pL/well of PBS-T.

[0069]

To 50 pL of serum was added 5 pL of PBS, and the mixture was gently stirred with vortex and allowed to stand for 5 minutes. Three times the volume of 1% BSA/PBS

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2018229466 12 Sep 2018 was added to this sample to dilute the serum. Fifty pL of the diluted sample was added to the capture antibody solid phase plate and shaken at 400 rpm at 37°C for 2 hours. After the reaction, the plate was washed three times with 300 pL/well of PBS-T.

[0070]

The ALP-labeled detection antibody was diluted 2400-fold with 1% BSA/PBS, 50 pL was added into each well, and shaken at 400 rpm at 37°C for 1.5 hours. After the reaction, the plate was washed three times with 300 pL/well of PBS-T.

100 pL/well of CDP-star was added to the plate, and allowed to react at room temperature for 10 minutes, and the luminescence intensity was measured.

[0071]

4-5. Results

The results are shown in Fig. 4. As is apparent from Fig. 4, the concentration of

OGFRL1 in the sera showed a marked increase in the mice administered with carbon tetrachloride compared with the non-injected control mice.

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Claims (13)

1. A method for determining a tissue regeneration state, comprising the steps of measuring opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject, comparing the measured OGFRL1 value with a reference value, and indicating that tissue regeneration is accelerated in the subject when the OGFRL1 value is equal to or greater than the reference value.

2. The method according to claim 1, wherein in the indication step, when it is indicated that the tissue regeneration is accelerated, it further indicates that the subject is in a recovery stage from disorder of the tissue.

3. The method according to claim 1 or 2, wherein the tissue is in an organ in which a non-neoplastic disease is present.

4. The method according to claim 3, wherein the organ is the liver.

5. The method according to claim 4, wherein the non-neoplastic disease present in the liver exhibits hepatic disorder.

6. The method according to any one of claims 1 to 5, wherein the subject is an individual having no malignant tumor in the liver.

7. The method according to any one of claims 1 to 6, wherein in the measurement

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2018229466 12 Sep 2018 step, OGFRL1 in serum, OGFRL1 in plasma, or OGFRL1 in extracellular vesicles present in a body fluid is detected.

8. A method comprising a step of detecting opioid growth factor receptor-like 1 (OGFRL1) in a body fluid collected from a subject, wherein an enhancement of tissue regeneration of the subject is indicated by the detection of OGFRL1.

9. The method according to claim 8, wherein the tissue is in an organ in which a non-neoplastic disease is present.

10. The method according to claim 9, wherein the organ is the liver.

11. The method according to claim 10, wherein the non-neoplastic disease present in the liver is hepatic disorder.

12. The method according to any one of claims 8 to 11, wherein the subject is an individual having no malignant tumor in the liver.

13. The method according to any one of claims 8 to 12, wherein OGFRL1 is detected in the serum, plasma, or extracellular vesicles present in a body fluid.

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Fig. 1 (MOLECULAR

WEIGHT) ^OGFRLI

B (LUMINESCENCE INTENSITY)

CARBON DEN-INDUCED LIVER

TETRACHLORIDE CANCER

ADMINISTRATION

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Fig. 2 (MOLECULAR

WEIGHT)

100,000 75,000 -

OGFRL1