JP2022059105A - Novel biomarker related to cancer - Google Patents

Abstract

To provide specification of the state of a subject who used a biomarker co-related to expression of FGF19.SOLUTION: In one embodiment, the present disclosure provides a biomarker co-related to expression of FGF19. In one embodiment, the biomarker of the present disclosure is protein. In one embodiment, the biomarker of the present disclosure is nucleic acid (for example, a cell-free DNA). In one embodiment, the present disclosure provides the use of the biomarker of the present disclosure as an indicator of the malignancy of such a proliferative disease as a cancer, an indicator of the effectiveness of an FGF19/FGFR4 route target medicine, or an indicator of the expression of FGF19, or provides the use of a FGF19/FGFR4 route target medicine.SELECTED DRAWING: None

Description

The present disclosure relates to novel biomarkers that can be used for applications such as indicators of malignancy of proliferative diseases such as cancer, indicators of efficacy of FGF19 / FGFR4 pathway targeting agents, indicators of expression of FGF19, and the like. The present disclosure also relates to novel uses of FGF19 / FGFR4 pathway targeting agents.

Fibroblast growth factor 19 (FGF19) is a hormonal protein expressed in humans and is known to be involved in bile acid biosynthesis, but it has also been suggested that it may be associated with tumors such as liver cancer (). Non-Patent Document 1). FGF19 also has the aspect of binding to fibroblast growth factor receptor 4 (FGFR4) and exerting its function via the FGF19 / FGFR4 signaling pathway. The in vivo functions of FGF19 and FGFR4 have not been completely elucidated, and there is a need for means that can be used directly or indirectly for their functional analysis.

Although several drugs targeting the FGF19 / FGFR4 pathway have been developed (Non-Patent Document 2), there may be subjects for which such drugs are effective and subjects for which such agents are not effective. Indicators for selection are sought.

Cells 2019, 8 (6), 536 Cancer Discov 2019; 9 (12): 1696-1707

The inventor has found that evaluation of FGF19 expression in cancer and the like can be difficult without direct measurement using tissue samples and the like, and further found a new biomarker that correlates with FGF19 expression. rice field. Based on this new finding, the present disclosure includes the use of this biomarker as an indicator of the malignancy of proliferative disorders such as cancer, the efficacy of FGF19 / FGFR4 pathway targeting agents, and the expression of FGF19. The use of FGF19 / FGFR4 pathway targeting drugs is provided.

Accordingly, the present disclosure provides:
(Item 1)
A method in which the expression of a biomarker selected from the biomarker of group (A) and the biomarker of group (B) in a subject having a proliferative disease is used as an index of the malignancy of the proliferative disease.
Including the step of measuring the expression of the biomarker in the sample derived from the subject.
The expression level of the biomarker selected from the measured biomarkers in the group (A) exceeds the reference value, or the expression level of the biomarker selected from the measured biomarkers in the group (B) exceeds the reference value. Below, it indicates that the proliferative disorder in the subject is likely to be highly malignant.
Method.
(Item 2)
A method in which the expression of a biomarker selected from the biomarker of group (A) and the biomarker of group (B) in a subject having a disease is used as an index of the efficacy of a FGF19 / FGFR4 pathway target drug.
Including the step of measuring the expression of the biomarker in the sample derived from the subject.
The expression level of the biomarker selected from the measured biomarkers in the group (A) exceeds the reference value, or the expression level of the biomarker selected from the measured biomarkers in the group (B) exceeds the reference value. Below, the subject is indicated to be an effective group against the FGF19 / FGFR4 pathway targeting agent.
Method.
(Item 3)
The method of any of the above items, wherein the sample comprises a blood sample.
(Item 4)
The method of any of the above items, wherein the biomarker is selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1 and SERPINI1.
(Item 5)
The method of any of the above items, wherein the biomarker is ST6GAL1.
(Item 6)
The method of any of the above items, wherein the disease is cancer.
(Item 7)
The method of any of the above items, wherein the cancer comprises liver cancer, breast cancer, pancreatic cancer or colorectal cancer.
(Item 8)
The method of any of the above items, wherein the FGF19 / FGFR4 pathway target drug is an anticancer agent.
(Item 9)
The method of any of the above items, wherein the FGF19 / FGFR4 pathway target drug is selected from the group consisting of lenvatinib, physogatinib, FGF401, H3B-6527 and BLU9931.
(Item 10)
The method of any of the above items, wherein the FGF19 / FGFR4 pathway target drug comprises lenvatinib.
(Item 11)
A method for testing the expression of FGF19 in a subject, comprising the step of measuring the expression of a biomarker selected from the biomarker of group (A) and the biomarker of group (B) in a sample derived from the subject.
(Item 12)
The method of any of the above items, wherein a disease that abnormally expresses FGF19 is indicated based on the measured expression of the biomarker.
(Item 13)
The method of any of the above items, wherein the biomarker is selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1 and SERPINI1.
(Item 14)
The method of any of the above items, wherein the biomarker is ST6GAL1.
(Item 15)
A kit for use in any of the above methods, comprising the biomarker detector.

In the present disclosure, it is intended that the one or more features described above may be provided in combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those of skill in the art upon reading and understanding the following detailed description, as appropriate.

The present disclosure provides biomarkers that correlate with FGF19 expression. By using such a biomarker, it is possible to evaluate a condition such as cancer (for example, malignancy, drug susceptibility, etc.) without imposing an excessive burden on the patient.

An outline of stable isotope labeling (SILAC) -based cell proteomics and secretomic using amino acids in cell culture medium. Proteins showing more than 0.5-fold downregulation in FGF19 siRNA-transfected cells compared to NC siRNA-transfected cells were selected and compared between Hep3B cells and Huh7 cells. 60 proteins in the supernatant and 37 proteins in cell lysate were common among Hep3B cells and Huh7 cells. Six proteins identified in both supernatant and cellular is described. Hep3B and HuH7 cells were transfected with NC or FGF19 siRNA. It is the result of analyzing the mRNA levels of CTGF, CCL20, GDF15, ST6GAL1, ORM1 and SERPINI1 by qPCR 72 hours after transfection. N = 4; *, p <0.05 (for NC siRNA); AU represents an arbitrary unit, respectively. (A) Correlation of ST6GAL1 and GDF15 mRNA levels (n = 3) (left) and FGF19 mRNA levels in 9 liver cancer cell lines (n.s., not significant; AU, arbitrary unit). show. (B) Shows the correlation between FGF19 and ST6GAL1 mRNA levels (top) and the correlation between FGF19 and GDF15 mRNA levels (bottom) in 373 HCC patients enrolled in the TCGA database portal. 62 surgically resected HCC tissue sections were stained with FGF19 and sorted by FGF19 expression by the TNM staging system between the FGF19 high group (n = 15) and the FGF19 low group (n = 47). The tumor grade (A) and recurrence-free survival (RFS) (B) to be evaluated are shown. 62 surgically resected HCC tissue sections were stained with FGF19 and serum ST6GAL1 measured by ELISA between the FGF19 high group (n = 15) and the FGF19 low group (n = 47) classified based on FGF19 expression. Indicates the level. *, P <0.05. The receiver operating characteristic (ROC) curves for the diagnosis of FGF19 high HCC patients by serum ST6GAL1 levels are shown. AUC, area under the curve. The cutoff value for each ROC curve was determined by the J statistics of Youden (gray dots). The correlation between serum ST6GAL1 levels and tumor size in 79 HCC patients who underwent surgical resection (left) and the correlation between serum ST6GAL1 levels and tumor grade by the TNM staging system (right) are shown. .. The RFS time of HCC patients with high and low serum ST6GAL1 levels is shown. (A) 62 surgically resected HCC tissue sections were stained with FGF19 and measured by ELISA between the FGF19 high group (n = 15) and the FGF19 low group (n = 47) classified based on FGF19 expression. The serum FGF19 level was shown. n. s. , Not significant. (B) Recipient operating characteristic (ROC) curves for the diagnosis of FGF19 high HCC patients by serum FGF19 levels are shown. AUC, area under the curve. (A) Correlation between serum FGF19 levels and tumor size in 77 HCC patients undergoing surgical resection (left) and between serum FGF19 levels and tumor grade by the TNM staging system (right). ) Is shown. (B) Relapse-free time-to-live (RFS) of HCC patients with high and low levels of serum FGF19. n. s. , Not significant. The baseline serum ST6GAL1 levels measured by ELISA in 96 advanced HCC patients treated with either lenvatinib or sorafenib are shown. n = 31 (sorafenib), n = 65 (lenvatinib); ns, not significant. (A) Shows overall survival (OS) of 96 advanced HCC patients treated with either lenvatinib (n = 65) or sorafenib (n = 31) as assessed by the Kaplan-Meier method. (B) Lenvatinib (43) or sorafenib (43) in the serum ST6GAL1 low group (62) for the ST6GAL1 high and ST6GAL1 low groups classified based on the cut-off value of serum ST6GAL1 level to identify FGF19 propellant HCC. OS time of patients treated with any of 19) and OS time of patients treated with either lenvatinib (22) or sorafenib (12) in the serum ST6GAL1 high group (34) (right) ) Is shown.

Hereinafter, the present disclosure will be described with reference to the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise noted. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.

The definitions and / or basic technical contents of terms specifically used in the present specification will be described below as appropriate.

(Definition)
As used herein, "fibroblast growth factor 19 (FGF19)" is a hormonal protein that is involved in the suppression of bile acid biosynthesis through downregulation of CYP7A1 expression and stimulates glucose uptake in adipocytes. Is known. As an ortholog protein of human FGF19, FGF15 in mouse is known. It is known that the expression of FGF19 can be improved in tumors such as breast cancer and liver cancer. Also, as an association between FGF19 and other diseases, increased FGF19 levels were observed in the liver of patients with cholestasis, and decreased FGF19 levels were chronic diarrhea due to malabsorption of bile acids, metabolic syndrome, non-alcoholic fatty liver. , And it is known that it can be observed in patients with insulin resistance. A typical sequence of human FGF19 is a nucleic acid sequence: NM_005117.2 and an amino acid sequence: NP_005108.1.

As used herein, "fibroblast growth factor receptor 4 (FGFR4)" is known to be a receptor protein that recognizes FGF19. Therefore, the FGF19 / FGFR4 pathway refers to a signaling pathway involving FGF19 and FGFR4. Further, the FGF19 / FGFR4 pathway target drug refers to a drug that affects the FGF19 / FGFR4 pathway by directly or indirectly regulating (increasing or reducing) the expression and / or function of FGF19 and / or FGFR4. Isoform 1 (nucleic acid sequence: NM_213647.2, amino acid sequence: NP_998812.1), isoform 2 (nucleic acid sequence: NM_022963.3, amino acid sequence: NP_075252.2), isoform 3 (nucleic acid sequence:) are used as FGFR4 in humans. NM_001291980.1, amino acid sequence: NP_001278909.1) are typical.

As used herein, "β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1)" is known to be an enzyme that catalyzes glucosylation of proteins. In humans, isoform a (nucleic acid sequence: NM_173216.2, amino acid sequence: NP_775323.1), isoform b (nucleic acid sequence: NM_173217.2, amino acid sequence: NP_775324.1) and the like are typical.

Unless otherwise stated, reference to each protein herein includes not only the protein having the amino acid sequence described in a particular accession number (or the nucleic acid encoding it), but also its functional equivalents. It is understood that it will be done.

As used herein, a "functional equivalent" of a molecule is a variant or variant of that molecule (eg, an amino acid sequence variant, etc.) and is a feature described herein (eg, a marker). ), Those that exhibit the same function (not necessarily the same degree) as the biological function of the molecule, and those that can change to the molecule itself at the time of action. Is understood to be included. As the functional equivalent of the present disclosure, an amino acid sequence in which one or more amino acids are inserted, substituted or deleted, or added to one or both ends thereof can be used.

As used herein, the "functional variant" of a molecule includes substances in which the molecule has been modified while maintaining (and to varying degrees) the function of the molecule. For example, a functional variant of a binding molecule, such as an antibody, is conjugated to another moiety (label, other functional molecule (protein, etc.), etc.) to maintain its binding function to the target molecule. , Fragmented ones, etc. are included. Thus, as used herein, a functional variant retains its underlying functionality prior to modification.

As used herein, the term "biological function" refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto. As used herein, biological function can be exerted by "biological activity". As used herein, "biological activity" refers to the activity that a factor (eg, polynucleotide, protein, etc.) can have in vivo, eg, another molecule by interaction with one molecule. Also includes activities that activate or inactivate. When two factors interact, their biological activity can be determined by the binding between the two molecules and the resulting biological changes, for example, one molecule is precipitated with an antibody. When other molecules are co-precipitated when they are allowed to do so, the two molecules are considered to be bound. For example, if a factor is an enzyme, its biological activity comprises that enzymatic activity. In another example, if a factor is a ligand, the ligand involves binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.

"Derivatives" or "similars" or "variants" of proteins or nucleic acids used herein are not intended to be limiting, but include molecules containing regions that are substantially homologous to the protein or nucleic acid. Such molecules, in various embodiments, are at least 30% compared to sequences aligned over identical size amino acid or nucleic acid sequences or aligned by computer homology programs known in the art. , 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical, or "derivatives" or "similars" or "variants" of such nucleic acids. Can be hybridized to the original nucleic acid under stringent, moderately stringent, or non-stringent conditions. Typically, a protein "derivative" or "similar" or "mutant" is a product of a naturally occurring protein that has undergone modifications such as amino acid substitutions, deletions, and additions, and is still an organism of the naturally occurring protein. Means a protein that exhibits scientific function, but not necessarily to the same degree. For example, it is also possible to investigate the biological function of such proteins by appropriate and available in vitro assays described herein or known in the art. Although this disclosure mainly discusses humans, it is understood that it also applies to other species, such as other species within primates or species of animals of other genera, and these mammals are also referred to in this book. It is understood that it falls within the scope of disclosure.

As used herein, a "functionally active" protein, polypeptide, fragment or derivative has a structural, regulatory, or biochemical function of the protein, such as biological activity.

As used herein, the term "gene" refers to a factor that defines a genetic trait. It is usually arranged in a certain order on the chromosome. A gene that regulates the primary structure of a protein is called a structural gene, and a gene that influences its expression is called a regulatory gene. As used herein, "gene" may refer to "polynucleotide", "oligonucleotide" and "nucleic acid" (here, including DNA, RNA, etc.). The "gene product" is a substance produced based on a gene and refers to a protein, mRNA or the like. Therefore, mRNA can be included in the concept of genes and can also be a gene product. In addition, "gene expression" often refers to transcriptional levels such as mRNA.

As used herein, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched or cyclic. The amino acid may be natural or non-natural, or may be a modified amino acid. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids), peptide-like compounds (eg, peptoids) and other modifications known in the art. Will be done.

As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length.

As used herein, "homology" of a gene means the degree of identity of two or more gene sequences to each other, and generally, having "homology" means a high degree of identity or similarity. say. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%. , 95%, 96%, 97%, 98% or 99% are homologous. Thus, as used herein, a "homologous" or "homologous gene product" is a protein in another species, preferably a mammal, that exerts the same biological functions as the protein components of the complex further described herein. Means. Such homologues are also sometimes referred to as "ortholog gene products."

Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code. As used herein, comparisons of amino acid and base sequence similarity, identity and homology are calculated using default parameters using BLAST, a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.2.9 (issued May 12, 2004). The value of identity in the present specification is usually the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is used as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity.

As used herein, a "purified" substance or biological factor (eg, nucleic acid or protein) is the removal of at least a portion of the factors naturally associated with the biological factor. Therefore, the purity of the biological factor in the purified biological factor is usually higher (ie, enriched) than in the state in which the biological factor is normally present. As used herein, the term "purified" is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. It means that the same type of biological factor is present. The substance used in the present disclosure is preferably a "purified" substance.

As used herein, a "corresponding" amino acid or nucleic acid has, or has, in a polypeptide molecule or polynucleotide molecule the same action as a given amino acid or nucleotide in a polypeptide or polynucleotide that serves as a reference for comparison. Amino acids or nucleotides that are expected to be predicted, especially in the case of enzyme molecules, amino acids that are present at similar positions in the active site and make similar contributions to catalytic activity. For example, if it is an antisense molecule, it can be a similar part in the ortholog corresponding to a particular part of the antisense molecule. The corresponding amino acids are specified to be, for example, cysteineized, glutathioneified, SS bond formation, oxidation (eg, oxidation of the methionine side chain), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Can be an amino acid. Alternatively, the corresponding amino acid can be the amino acid responsible for dimerization. Such "corresponding" amino acids or nucleic acids may be regions or domains over a range. Thus, such cases are referred to herein as "corresponding" regions or domains.

As used herein, the term "expression" of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo and becomes another form. Preferably, a gene, polynucleotide, or the like is transcribed and translated into the form of a polypeptide, but transcribed to produce mRNA may also be an aspect of expression. More preferably, the form of such a polypeptide can be post-translationally processed (derivatives as used herein). For example, the expression level of a molecule can be determined by any method. Specifically, the expression level can be known by assessing the amount of mRNA, the amount of protein, or the biological activity of the protein in this molecule. Such expression levels are evaluated using the antibodies of the present disclosure by any suitable method including immunological measurement methods such as ELISA method, RIA method, fluorescent antibody method, Western blotting method, immunohistochemical staining method and the like. The poly used in the present disclosure to be evaluated by any suitable method, including expression levels of the disclosed polypeptides at the protein level, or molecular biological measurements such as Northern blotting, dot blotting, PCR. The expression level of the peptide at the mRNA level can be mentioned. "Change in expression" is the expression of the polypeptide used in the present disclosure at the protein or mRNA level as assessed by any suitable method, including the immunological or molecular biological measurement methods described above. It means that the amount increases or decreases. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.

As used herein, the term "marker (substance, protein or gene (nucleic acid))" refers to a substance that serves as a marker for tracking whether a condition (eg, a disease) is present or at risk thereof. Examples of such markers include genes (nucleic acid = DNA level), gene products (mRNA, proteins, etc.), metabolites, and the like. In the present disclosure, detection, diagnosis, preliminary detection, prediction or pre-diagnosis of a condition is a drug, agent, factor or means specific to the marker associated with the condition, or a composition, kit or system containing them. It can be realized by using such as.

As used herein, the term "treatment" means to prevent, preferably to maintain the status quo, more preferably to alleviate, and even more preferably to eliminate the exacerbation of a disease in the event of such a condition. This includes the ability to exert a symptom-improving effect or a preventive effect on a patient's disease or one or more symptoms associated with the disease. Diagnosis in advance and appropriate treatment is called "companion treatment", and the diagnostic agent for that purpose is sometimes called "companion diagnostic agent".

As used herein, the term "prevention" refers to preventing a disease from becoming such a condition before it becomes such a condition.

As used herein, the term "diagnosis" refers to identifying various parameters related to a condition (eg, disease) in a subject and determining the current state or future of such condition. By using the methods, compositions, and systems of the present disclosure, the condition within the body can be investigated and such information can be used to determine the condition in the subject, the procedure or method for administration, etc. Various parameters can be selected. In the present specification, "diagnosis" in a narrow sense means diagnosing the current state, but broadly includes "early diagnosis", "predictive diagnosis", "pre-diagnosis" and the like. As a general rule, the diagnostic method of the present disclosure is industrially useful because it can be used from the body and can be carried out without the hands of medical personnel such as doctors. In the present specification, "predictive diagnosis, pre-diagnosis or diagnosis" may be particularly referred to as "support" in order to clarify that it can be carried out without the hands of medical personnel such as doctors. The technique of the present disclosure can be applied to such a diagnostic technique.

As used herein, "antibody" is broadly referred to as a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a chimeric antibody, and an anti-idiotype antibody, and fragments thereof, such as an Fv fragment, a Fab'fragment, and an F (ab'). ₂ and Fab fragments, as well as conjugates or functional equivalents produced by other recombination (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chains. Includes antibodies, scFV, diabodies, sc (Fv) ₂ (single chain (Fv) ₂ ), scFv-Fc). Further, such an antibody may be covalently linked or recombinantly fused to an enzyme such as alkaline phosphatase, horseradish peroxidase, alpha-galactosidase, or the like. The origin, type, shape, etc. of the antibody used in the present disclosure are not limited. Specifically, known antibodies such as non-human animal antibodies (eg, mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, humanized antibodies and the like can be used. In the present disclosure, monoclonal or polyclonal can be used as an antibody, but a monoclonal antibody is preferable. The binding of the antibody to the target protein is preferably a specific binding.

As used herein, the term "means" refers to any tool that can be an arbitrary tool for achieving a certain purpose (eg, detection, diagnosis, treatment, prevention, migration), and in particular, in the present specification, "selective recognition (selective recognition (eg)". "Means for detecting" means means for recognizing (detecting) a certain object differently from others.

As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression uses appropriate methods, including, for example, mRNA measurement and immunological measurement methods, including binding or interaction with marker detectors. Can be achieved. Examples of the molecular biological measurement method include Northern blotting, dot blotting, PCR and the like. Examples of immunological measurement methods include ELISA method using a microtiter plate, RIA method, immunofluorescence method, luminescence immunoassay (LIA), immunoprecipitation method (IP), immunodiffusion method (SRID), and immunity. Examples thereof include immunofluorescence (TIA), Western blotting, and immunohistochemical staining. Moreover, as a quantification method, an ELISA method, an RIA method and the like are exemplified. It can also be performed by a gene analysis method using an array (for example, a DNA array or a protein array). DNA arrays are widely outlined in (Shujunsha ed., Cell Engineering Separate Volume "DNA Microarrays and Latest PCR Methods"). For protein arrays, see Nat Genet. It is detailed in 2002 Dec; 32 Suppl: 526-32. Examples of the method for analyzing gene expression include, but are not limited to, RT-PCR, RACE method, SCSP method, immunoprecipitation method, two-hybrid system, in vitro translation, and the like. Such further analytical methods are described, for example, in the Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, edited by Yusuke Nakamura Yodosha (2002), and all of these descriptions are incorporated herein by reference. Will be done.

The detection agent or detection means of the present disclosure may be a complex or a complex molecule in which another substance (for example, a label or the like) is bound to a detectable portion (for example, an antibody or the like). As used herein, "complex" or "complex molecule" means any construct that comprises two or more moieties. For example, if one portion is a polypeptide, the other portion may be a polypeptide or other substance (eg, substrate, sugar, lipid, nucleic acid, other hydrocarbon, etc.). May be. In the present specification, two or more portions constituting the complex may be bonded by covalent bonds or other bonds (for example, hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals forces, etc.). It may have been done. When two or more portions are polypeptides, it can also be referred to as a chimeric polypeptide. Therefore, as used herein, the term "complex" includes molecules formed by linking multiple types of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules.

As used herein, the term "probe" refers to a searchable substance used in biological experiments such as in vitro and / or in vitro screening, for example, a nucleic acid molecule containing a specific base sequence or a specific. Examples include, but are not limited to, peptides containing amino acid sequences, specific antibodies or fragments thereof. In the present specification, the probe is used as a marker detection means.

As used herein, the term "label" refers to an entity (eg, substance, energy, electromagnetic wave, etc.) for identifying a molecule or substance of interest from others. Examples of such a labeling method include an RI (radioisotope) method, a fluorescence method, a biotin method, a chemiluminescence method and the like. When a plurality of markers or factors or means for capturing the markers of the present disclosure are labeled by a fluorescence method, the markers are labeled with fluorescent substances having different fluorescence maximum wavelengths. The difference in the maximum wavelength of fluorescence emission is preferably 10 nm or more. When labeling the ligand, any one that does not affect the function can be used, but Alexa ^TM Fluor is desirable as the fluorescent substance. Alexa ^TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that supports a wide range of fluorescent wavelengths. It is stable, bright, and has low pH sensitivity. Examples of the combination of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa ^TM 555 and Alexa ^TM 633, a combination of Alexa ^TM 488 and Alexa ^TM 555, and the like. When labeling a nucleic acid, any one that can bind to the base portion thereof can be used, but cyanine dyes (for example, Cy3, Cy5, etc. of CyDye ^TM series), Rhodamine 6G reagent, N-acetoxy-N2- It is preferable to use acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like. Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and Rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of Rhodamine 6G reagent and fluorescein, and the like. In the present disclosure, such a label can be used to modify an object of interest so that it can be detected by the detection means used. Such modifications are known in the art and those skilled in the art can optionally implement such methods depending on the label and the subject of interest.

As used herein, a "tag" is a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a binding for binding a specific substance. A substance that acts as a partner (eg, having a relationship such as biotin-avidin, biotin-streptavidin) and may be included in the category of "label". Thus, for example, a specific substance to which a tag is bound can be selected by contacting the base material to which the binding partner of the tag sequence is bound. Such tags or signs are well known in the art. Typical tag sequences include, but are not limited to, myc tags, His tags, HA, Avi tags, and the like. Such tags may be attached to the markers or marker detectors of the present disclosure.

As used herein, "drug", "agent" or "factor" (both correspond to agents in English) are broadly used interchangeably as long as they can achieve their intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity). Such substances include, for example, cells (eg, T cells), proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, cDNA, DNA such as genomic DNA, mRNA. Such as RNA), polypeptides, oligosaccharides, lipids, organic small molecules (eg, hormones, ligands, signaling substances, organic small molecules, molecules synthesized with combinatorial chemistries, small molecules that can be used as pharmaceuticals (eg, etc.). , Small molecule ligands, etc.), etc.), these complex molecules include, but are not limited to.

As used herein, the term "kit" refers to a unit that is usually divided into two or more sections and is provided with parts to be provided (for example, a diagnostic agent, a therapeutic agent, an instruction manual, etc.). The form of this kit is preferred when the purpose is to provide a composition such that it should not be mixed and provided for stability and the like, and it is preferable to mix and use immediately before use. Such kits preferably have instructions or instructions describing how to use the provided parts (eg, test, diagnostic, therapeutic) or how to treat the reagents. It is advantageous to have a book.

As used herein, the "instruction" describes the method of using the present disclosure to a physician or other user. This instruction sheet contains words instructing the detection method of the present disclosure, how to use a diagnostic agent, or administration of a drug or the like. In addition, the instruction sheet may include words indicating the administration site or administration method. This instruction is prepared and approved by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare in Japan, Food and Drug Administration (FDA) in the United States, etc.). It is clearly stated that it has been received. Instructions are so-called package inserts, usually provided in paper media, but are not limited to, for example, in the form of electronic media (eg, homepages provided on the Internet, e-mail). But can be provided.

The term "about" as used herein refers to the value shown plus or minus 10%. Even if it is not explicitly indicated as "about", it can be interpreted synonymously with "about".

(Preferable embodiment)
Hereinafter, preferred embodiments of the present disclosure will be described. It is understood that the embodiments provided below are provided for a better understanding of the present disclosure and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in consideration of the description in the present specification. It is also understood that the following embodiments of the present disclosure may be used alone or in combination.

In one aspect, the present disclosure provides biomarker-based identification (eg, diagnosis) of a subject's condition in which a correlation with FGF19 expression has been found. This can be achieved by any means, such as implementation of the method, provision of compositions, combinations, kits, or systems for this purpose. For example, even if there is no explicit description, the description of the method of using a certain biomarker for determining a certain state is based on the same biomarker detection agent for determining the same state and the same biomarker. Embodiments that reflect other means, such as methods of regulating the condition or agents thereof, are also contemplated.

(Biomarker that correlates with FGF19 expression)
In one embodiment, the disclosure provides a biomarker that correlates with the expression of FGF19, which may be referred to herein as the "biomarker of the present disclosure". The biomarkers of the present disclosure include the biomarkers of group (A) and the biomarkers of group (B).

A group of the following genes has been found as a biomarker that positively correlates with the expression of FGF19, and this group of biomarkers is referred to herein as the biomarker of group (A) or the gene of group (A).
Genes of group (A) A1BG, A2M, ABCC2, ABCF3, ABT1, ACSL4, ACSL5, ACTN3, ADAM10, ADCK3, AFM, AFP, AGR2, AGRN, AGT, AHR, AHSG, AKR1C2, ALDH16A1, ALG3 , ANG, ANGPTL3, ANKRD17, ANLN, ANXA1, APIP, APPP2, APOA2, APOC1, APP, ARID1B, ARMX1, ARSB, ASGR2, ASNS, ATP1A4, ATP2C1, ATP6AP1, ATP6AP2, ATP6AP1, ATP6AP2 , BPGM, BTD, C11orf98, C14orf1, C19orf80, C1GALT1C1, C2, C3, C4BPA, C5, C5orf42, C6, C8G, CAP1, CAP2, CASC3, CBR1 (CBR3), CCDC137, CCRCLC59, CCDC137, CDC25C, CDH1, CDK6, CDKL1 (CDKL3; CDKL2; PRKD2; PRKD3; CDKL5; STK36), CDO1, CEBPB, CETN2, CFI, CGGBP1, CHTF8, CIB1, CLN5, CLSTN1, CLSTN3, CLSTN1 , COX15, COX17, COX6B1, CP, CPB2, CPD, CPN1, CPN2, CPSF7, CPVL, CRLF1, CRLF3, CST3, CTGF, CTH, CTNNA2, CTSC, CUTC, CXADR, CXCL1, CXCL3 (CXCL2), CXCL8 CYHR1, DAB2, DCAF16, DDX5, DHFRL1, DIAPH3, DIEXF, DIMT1, DKK1, DMXL1, DNAH9, DNAJB11, DPH5, DSC2, DTNA, DTYMK, DYNLL2, EFNA1, EHBP1, EFBP1, EFBP1, EPB41L2, EPHA2, EPS8, ERCC6L2, EXTL2, EYA3, F13B, F2, F5, FAM134C, FAM169A, FAM3C, FAM73A, FAM96A, FBRN1, FBXL12, FBXO22, FEN1, FGA, FG FHOD1, FIBP, FKBP3, FLCN, FLRT3, FRAS1, FRRS1, FST, FTL, FUCA2, FURIN, FYTTD1, G3BP2, GAA, GABPA, GAK, GALNT2, GALNT4, GBA, GC, GCNT2, GCNT3 GLA, GLS, GM2A, GNPTG, GOLM1, GOLPH3L, GOT1, GPC1, GPR126, GPT2, GYG1, H2AFX, HAL, HBB, HEXA, HINT1, HLA-A, HMCES, HMGN2 (HMGN2) , ICAM1, IFI30, IFRD1, IFRD2, IGFBP3, INPP1, IPO13, IPO4, IREB2, ISOC1, ITGA2, JAG1, KALRN, KCNN4, KHNYN, KIF2B, KLC4, KMT2D, KNTC1, KYNLPL1 , LGMN, LIPC, LRG1, LSR, LUM, LYN, LYRM7, LYZ, MAFF (MAFK; MAFG), MAGI1, MAN1A1, MAN1B1, MAN2A1, MAP2K3, MASP1, MDC1, MDK, MEP1A, METTL MIPEP, MKRN2, MMAA, MMP15, MTAP, MTG2, MTHFD2, MTPP, NANS, NAT8, NDEL1 (NDE1), NGEF, NME1, NORC1, NOM1, NOMO2 (NOMO1; NOMO3), NPC2, NPTX2, NRXN3 NUCB2, OGFOD1, OLA1, ORM1, ORM2, OS9, OSBPL2, OTUD7B, PAM, PARD6G (PARD6A), PARS2, PCDHB3 (PCDHB8; PCDHB16; PCDHB7; PCDHB4; PDE4DIP (CDK5RAP2), PDGFD, PDGFRL, PDXP, PEX13, PFKM, PIAS1, PISD, PLA2G15, PLA2G7, PLD1, PLD3, PLG, PLOD3, PLS1, PON2, POP5, POP7, PPAN, PPTROM1 , PSAT1, PSM D7, PSMD9, PSME4, PTMS, PTP4A2, PTPN12, PTPRK, PTRH1, PVR, QSOX2, RAB21, RANBP2, RAVER1, RBBP6, RBL1, RBP4, RCOR1 (RCOR3), RFFL, RIOK2, RNF126, RN , RPL6, RPRD1A, RPS27L, RPS6KB2 (RPS6KB1), RPS8, SAA4, SCG2, SCPEP1, SCYL1, SDC1, SDC2, SDC4, SDCBP, SDF4, SEC24D, SEMA3A, SEMA4G, SEPT9, SERPINE1, SERPINI1, SGCE, SGK3, SH3GL1, SIAE, SIL1, SLAIN1, SLC1A4, SLC1A5, SLC38A1, SLC39A10, SLC39A14, SLC3A2, SLC6A11, SLC7A1 SPAG5, SPCS1, SPINK1, SPOCK2, SPON2, SPP1, SPTB, SPTN4, SQSTM1, SRSF3, ST6GAL1, STC2, STK32C, STK4, STRIP2, STX18, STX8, STXBP5, SUGP2, SULT1 , TARS2, TBC1D8B, TBC1D9, TFPI, TFRC, TGOLN2, THBS1, THBS4, THOC7, TMBIM6, TMCO5A, TMEM109, TMF1, TMPO, TMPRSS13, TNFRSF11B, TOP3B, TOR1B , TSPYL1, TSPYL2, TTR, TUBE1, UBA52, UBAC2, UBE2B, UBE2S, UBE2T, UBE3C, UBFD1, UBL5, UBL7, UPF1, URB2, UXS1, VAMP3 (VAMP2) WDR55, WRAP53, YIPF4, ZC3H13

The following groups of genes have been found as biomarkers that negatively correlate with FGF19 expression in multiple cell lines, and this group of biomarkers is referred to herein as the biomarker of group (B) or of group (B). Called a gene.
-Genes of group (B) ABI3BP, ACE, ACHE, ACTA1 (ACTC1; ACTG2; ACTA2), ACTG1, ACTN1, ACTN2, ADAMTS13, AIFM1, ALAD, ALCAM, ALDH9A1, ALDOB, ALYREF, ANPEP, AOC3 APMAP, APOC3, ARPT, ARF1 (ARF3; ARF5), ARHGDIB, ARMT1, ARPC4, BHMT, C1QTNF3, C3P1, C4A (C4B), C8A, C8B, CAB39, CACNA2D1, CADM1, CAMK2D CAPRIN1, CAT, CBRN4, CBS, CBX5, CCT2, CCT3, CCT5, CD109, CD276, CD44, CDC42, CDH11, CDH13, CDH5, CDH6, CFD, CHAD, CHST3, CILP2, CLIC11A, CLIC4, CNN2, CNTFR CNTRL, COL11A1, COL11A2, COL12A1, COL18A1, COL1A1, COL1A2, COL21A1, COL2A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL9A1, COMP, COPY CREG1, CROCC, CSPG4, CSTB, CUL1, CUTA, CYCS, DCD, DCUN1D1, DDAH2, DDX42, DDX6, DEK, DKK3, DMBT1, DPYS, DSC1, DSG1, DSP, DYNC1I2, EEMP2 EIF3A, EIF3F, EIF3M, EIF4A1, EIF4A3, EML2, ENOPH1, ENPP1, ERAP1, ERP29, EWSR1, EXOSC9, EXT1, EZR, F10, F11, F13A1, F9, FAH, FAP, FAT4, FB1 FLNA, FLNB, FLNC, FMOD, FSCN1, FSTL1, FUBP1, G6PD, GCLC, GCLM, GDI1, GDI2, GGCT, GLIPR2, GLO1, GRHPR, GSN, GSS, GSTM2, GSTM3, H2AFSY3 HI ST1H2AJ (HIST1H2AH; H2AFJ; HIST2H2AC; HIST2H2AA3; HIST1H2AD; HIST1H2AG), HIST1H4A, HIST2H3A, HMCN2, HNRNPR, HNRNPUL2, HPD, HP , IMPDH2, IPO9, IQGAP1, ITGB1, JUP, KARS, KATNAL2, KLKB1, KPNA2, KPNA3, KPNA4, KPRP, KRT18, LAMA2, LAMB1, LCAT, LCP1, LDHB, LECT2, LGLTALS3 , LTDBP4, LTD, LUC7L2 (LUC7L), MAGOHB (MAGOH), MAPRE2, MCM2, MCM3, MFAP4, MGAT2, MMP2, MRC2, MSN, MST1, MYH10, MYH9, MYL6, MYLK, NACAP1 , NELL2, NEO1, NES, NME2, NONO, NOP58, NOTCH1, NOTCH2, NOTCH3, NPM1, NRP2, NT5C2, NUTF2, OGN, OMD, P4HB, PAIP1, PARP1, PARVB, PCDH17, PCDH18, PCDH18 , PDIA6, PEPD, PFKL, PGAM4, PGM1, PGM2, PKHD1L1, PLRG1, PLS3, PLXDC2, PLXNA1, POSTN, PPP1CB, PPP1R12A, PPP2R4, PPP4C, PPP6C, PRDX2, PRDX4 PSMA1, PSMA3, PSMA4, PSMB1, PSMB3, PSMB4, PSMB5, PSMC5, PSMC6, PSMD14, PSMD6, PSMD8, PSME1, PTPN11, PTPRD, PTPRG, PTPRM, PTPRS, PVRL1, PWP1, PYGB, PYGL, RAP1 , RCN1, RCN3, RDX, RGN, RPLP2, RPS15A, RPS6KA3, RPSA, RSF1, RSU1, RUVBL2, S100A1, S100A12, S100A7, S100 A8, S100A9, SAFB, SAR1B (SAR1A), SART3, SEC23A, SEC24C, SEP11, SEPT2, SEPT7, SEPT9, SERPINB1, SERPINH1, SF3B3, SH3BGRL3, SMARCA1, SMARCC1, SMS1, SPRN , SPP2, SRRM1, SRSF1, SRSF5, SSRP1, TAGLN2, TCP1, TECPR2, TG, TGFBR3, THBS3, THRAP3, THUMPD1, TIE1, TLN1, TMEM132C, TNC, TNXB, TPM1, TPM3 , TSPYL2, TUBA4A, TWF2, TXNDC5, TXNL1, UBA3, UBE2K, UBE2L3, UBE2NL, UBE2V1 (UBE2V2), UGP2, VASN, VAT1, VCAM1, VCL, VCP, VN1 XRN2, YWHAE, YWHAH, ABHD17A, ACSS2, AHANAK, ANGPTL3, ANKRD46, ANO10, AP1G2, APOA1, APOC3, APOE, ASRGL1, BIN1, C3orf58, CASK, CDS2, CHML, CTP EEF1A1 (EEF1A1P5), ENO3, EPB41L3, EPHX1, EPPC1, EPS8L2, FDPS, FTL, FUCA1, FUNDC2, GALNT1, GCN1L1, GLUD1, GM2A, GNG5, GPX7, TP KIAA1161, KLHL25, L3HYPDH, LPCAT1, LSM14A, MANBA, MFGE8, NDRG1, NDUFS4, NHLRC3, NID1, NPEPL1, NT5DC2, OPA3, OPLAH, P4HA2, PALMD, PBX1PL2 PPL, PPM1A, PPM1H, PPP1R18, PRKAR2A, PRRC2B, QKI, QSOX1, RBP1, REEP6, RELL1, S100A10, SAMHD1, SAR1 B, SCAMP1, SEC24A, SERPINA5, SERPINF2, SFN, SLC12A2, SLC30A10, SLC46A1, SNAP25, SNX27, STATS2L, STAT3, SWAP70, TCAF1, TGFBI, TLN2, TM7SF2, TMEM177, TMEM2 TUBB2B, TUBB8, VTN, ZFYVE19

As the biomarkers of the present disclosure, among the biomarkers of group (A), the following genes are commonly found in the secretome of a plurality of cell lines and may be particularly preferably used: A1BG, A2M, ACSL4, AGRN. , AHSG, APPL2, APOA2, APOC1, APP, ARID1B, ATP6AP2, B3GAT2, CCL15, CCL20, CLSTN3, CLU, CP, CTGF, DNAJB11, EXTL2, F2, F5, FGA, FGL1, FST, FUG15 , GPR126, HNRNPA3, ICAM1, LDLR, LGALS3BP, LRG1, LYZ, MAN1A1, MEP1A, NTS, ORM1, PCOLCE2, PPT1, PROS1, SDC4, SDCBP, SERPINA10, SERPINA3, SERPINS1 , TFRC, TNFRSF11B, TOR1B, TTR, UXS1, VWA1. As the biomarkers of the present disclosure, among the biomarkers of group (A), the following genes are commonly found in whole proteomes of a plurality of cell lines and may be particularly preferably used: ANLN, ANXA1, ARMCX1, ATP1A4, CCL20, CTGF, DYNLL2, FIBP, FYTTD1, GDF15, H2AFX, HMCES, HSPA13, IFRD1, INPP1, IREB2, ITGA2, MAP2K3, NT5E, ORM1, PARS2, PSME4, SMG6, ST6GAL1, SUGP2, THBS4, TMPO, TUBE1, UBE2B, VAMP3 (VAMP2), YIPF4. As the biomarkers of the present disclosure, among the biomarkers of group (A), the following genes are commonly found in whole proteomes and secretomes of multiple cell lines and may be used more preferably: CTGF, CCL20, GDF15, ST6GAL1, ORM1, SERPINI1. In the most preferred embodiment, the biomarker of the present disclosure is ST6GAL1.

As the biomarkers of the present disclosure, among the biomarkers of group (B), the following genes are commonly found in whole proteomes and secretomes of a plurality of cell lines and may be used more preferably: ACTG1, ALDH9A1, etc. ANPEP, CDH6, COL18A1, DCD, FLNC, FSCN1, GSTM2, GSTM3, HIST1H4A, HIST2H3A, HPX, HSPB1, IL1RAP, S100A7, TLN1, YWHAH.

In one embodiment, the biomarker that correlates with FGF19 expression is one or more genes selected from group (A) and group (B) or a transcription or translation product thereof. In one embodiment, the biomarker that correlates with FGF19 expression is a gene selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1, SERPINI1 or a transcription or translation product thereof. These proteins have been shown to correlate with FGF19 expression at both intracellular and culture supernatant expression levels. In a particularly preferred embodiment, the biomarker that correlates with FGF19 expression is the ST6GAL1 gene or a transcription or translation product thereof. Serum ST6GAL1 levels were found to correlate with FGF19 expression and robustness in tumor tissues and are good biomarkers of tumor drug susceptibility. In one embodiment, the biomarker of the present disclosure is a protein. In one embodiment, the biomarker of the present disclosure is a nucleic acid (eg, cell-free DNA).

In one embodiment, the biomarkers of the present disclosure may be assessed at expression levels in a subject or in a sample obtained from a subject. In one embodiment, the biomarkers of the present disclosure can be assessed by the level of expression (eg, the amount of secreted protein, cell-free DNA) in blood or blood sample (eg, serum sample). The inventors have found that FGF19 levels in blood are less correlated with FGF19 levels in tissues at diseased (eg, cancer) sites, in contrast to the biomarkers of the present disclosure in blood (particularly, in particular). It was unexpectedly found that the level of ST6GAL1) was highly correlated with the FGF19 level in the tissue at the diseased (eg, cancer) site and with the malignancy of the disease. Therefore, the identification of the subject's condition using the biomarkers of the present disclosure can be carried out without imposing a large load on the subject.

(Identification of subject's condition based on biomarkers)
In one aspect, the present disclosure provides identification (eg, diagnosis) of a subject's condition with the biomarkers of the present disclosure as indicators. In one embodiment, the subject's condition identified includes the presence or absence of a disease in the subject, the condition of the disease in the subject (malignancy, stage, etc.), the risk of the disease in the subject, the drug sensitivity of the subject, and the like. Can be mentioned. The biomarkers of the present disclosure can correlate with FGF19 expression and thus due to any disease that may be associated with FGF19 expression, such as proliferative disorders such as cancer, cholestasis, bile acid synthesis abnormalities, bile acid malabsorption. It can be used to identify conditions such as chronic diarrhea, metabolic syndrome, non-alcoholic fatty liver, and insulin resistance. In one embodiment, the proliferative disorders whose status can be identified by the biomarkers of the present disclosure are cancers (eg, liver cancer, breast cancer) in which high expression of FGF19 is associated with high malignancy thereof. obtain. High malignancy of a cancer can mean that the cancer is likely to relapse, has a poor prognosis, and / or is metastatic. In one embodiment, the biomarkers of the present disclosure can be used to identify the malignancy of proliferative disorders. In one embodiment, the biomarkers of the present disclosure can be used to identify the stage of proliferative disease. Although the biomarkers of the present disclosure have been found by correlating with the expression of FGF19, they do not necessarily have to be associated with the expression of FGF19 and are described herein using only the biomarkers of the present disclosure as indicators. The condition can be specified (for example, diagnosis). In one embodiment, a subject attempting to identify a condition using the biomarkers of the present disclosure has been diagnosed with a disease (eg, cancer).

Proliferative disorders whose condition (eg, malignancy, drug susceptibility) can be identified by the biomarkers of the present disclosure include breast cancer, prostate cancer, pancreatic cancer, gastric cancer, lung cancer, colon cancer (colon cancer, rectal cancer, anal). Cancer), esophageal cancer, duodenal cancer, head and neck cancer (tongue cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer), brain tumor, nerve sheath tumor, neuroblastoma, glioma, non-small cell lung cancer, small cell lung cancer, liver Cancer, kidney cancer, bile duct cancer, uterine cancer (uterobody cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphoma, malignant melanoma, bone tumor, vascular fibroma, retinal sarcoma, penis Cancer, Pediatric solid cancer, Kaposi's sarcoma, maxillary sinus tumor, fibrous histiocytoma, smooth muscle tumor, rhizome myoma, liposarcoma, uterine myoma, osteoblastoma, osteosarcoma, chondrosarcoma, mesophageal tumor, leukemia And so on. In certain embodiments, the proliferative disorder whose condition can be identified by the biomarkers of the present disclosure can be liver cancer.

In one embodiment, the biomarkers of the present disclosure can be used to identify drug susceptibility of a subject having a disease (eg, cancer). In one embodiment, the biomarkers of the present disclosure can correlate with FGF19 expression and can be used to identify subjects for whom the FGF19 / FGFR4 pathway targeting agent is effective. FGF19 / FGFR4 pathway targeting agents refer to agents that regulate the function or expression of any biomolecule (mRNA, protein, etc.) in the signaling pathways involving FGF19 and FGFR4. In one embodiment, the FGF19 / FGFR4 pathway targeting agent regulates the function or expression of FGF19 and / or FGFR4. In one embodiment, the FGF19 / FGFR4 pathway target drug is an inhibitor of the FGF19 / FGFR4 pathway. In one embodiment, the FGF19 / FGFR4 pathway target drug reduces the expression of FGF19 or FGFR4. In one embodiment, the FGF19 / FGFR4 pathway target drug inhibits the binding of FGF19 to FGFR4. In one embodiment, the FGF19 / FGFR4 pathway targeting agent inhibits the kinase activity of FGFR4, where the inhibition may be FGFR4 selective or in addition to FGFR4 other FGFRs (eg, FGFR1, FGFR2 and / or FGFR3) may be inhibited. In one embodiment, the FGF19 / FGFR4 pathway target drug is an anticancer agent. In one embodiment, the FGF19 / FGFR4 pathway targeting agent is a small molecule compound, protein (eg, any protein in the FGF19 / FGFR4 pathway, or a functional variant thereof), antibody, or nucleic acid (eg, FGF19 / FGFR4 pathway). Nucleic acid that suppresses mRNA encoding any of the proteins in. For example, small molecule compounds such as lenvatinib, physogatinib (Cancer Discov 2019; 9 (12): 1696-1707), FGF401 (Mol Cancer Ther; 18 (12) December 2019), H3B- 6527 (Cancer Res; 77 (24) December 15, 2017) and BLU9931 (Cancer Discov. 2015 Apr; 5 (4): 424-37), but not limited to one of the above functions. Any known small molecule compound having one or more can be used.

In one embodiment, the biomarkers of the present disclosure can be used to identify the expression level of FGF19 (eg, tissue expression level) in a subject. Since the biomarkers of the present disclosure can be alternative markers for tissue expression of FGF19, they can be used for any purpose such as research purposes.

In one embodiment, the biomarkers of the present disclosure may be combined with other indicators. In one embodiment, the biomarkers of the present disclosure may be used to identify the condition of a subject and then the expression level of FGF19 (eg, tissue expression level) of this subject.

(Reference value of biomarker)
In one embodiment, a subject is identified (eg, diagnosed) as having a particular condition if the expression level of the biomarkers of the present disclosure is outside a predetermined range. A predetermined range of biomarkers of the present disclosure may vary depending on the type of biomarker used and the type of condition of the subject to be identified, and those skilled in the art will use the predetermined range of biomarkers of the present disclosure. A specific numerical range can be set appropriately. In one embodiment, the biomarkers of group (A) of the disclosed biomarkers identify a subject as having a particular condition (eg, diagnosis) when its expression level exceeds a predetermined reference value. ). In one embodiment, the biomarkers of group (B) of the disclosed biomarkers are identified as having a particular condition (eg, diagnosis) if their expression level is below a predetermined reference value. ). In one embodiment, a predetermined range of biomarkers of the present disclosure can be determined based on the correlation between the biomarker and the presence or absence of the condition of the subject to be identified. For example, when subjects are divided into two groups according to the presence or absence of a condition (presence or absence of disease, presence or absence of drug sensitivity, presence or absence of FGF19 expression in tissues exceeding a predetermined level, etc.), specific sensitivity, specificity, and specificity, and / Or the expression level of the biomarkers of the present disclosure that achieves a false positive rate can be set as a reference value. In one embodiment, the defined range of biomarkers of the present disclosure is specific sensitivity when subjects with a particular disease (eg, cancer) are divided into two groups based on FGF19 expression levels in lesioned tissue. It is set to achieve specificity and / or false positive rate. By making a determination based on the reference values of the biomarkers of the present disclosure thus set, subjects with a disease (eg, cancer) can be further stratified and more appropriate treatment (eg, drug selection). ) Can be provided. In one embodiment, the reference value for the expression level of the biomarkers of the present disclosure can be determined by the Youden index, and a ROC curve is created for each cutoff value to generate a true positive rate (TRP) -false positive rate (TRP) -false positive rate ( FRP) can be calculated and the cutoff value that maximizes the true positive rate (TRP) -false positive rate (FRP) can be selected as the reference value.

In one embodiment, the criteria for identifying subjects with FGF19 / FGFR4 pathway target drug sensitive and / or high-grade cancer using serum ST6GAL1 levels are about 15 ng / ml or more. About 16 ng / ml or more, about 17 ng / ml or more, about 18 ng / ml or more, about 19 ng / ml or more, about 20 ng / ml or more, about 21 ng / ml or more, about 22 ng / ml or more, about 23 ng / ml or more, about 24 ng It can be at serum ST6GAL1 levels greater than or equal to / ml, or greater than or equal to about 25 ng / ml, and in particular embodiments greater than or equal to about 19.1 ng / ml.

(Measurement of biomarkers)
Each of the biomarkers of the present disclosure can be detected using any suitable detection agent, and the expression level can be measured. In one embodiment, the detection agent is a molecule that specifically binds to the biomarkers of the present disclosure, eg, an antibody, a protein or nucleic acid known to bind in vivo, a complementary nucleic acid, or a small molecule compound. possible. In one embodiment, the detection agent may include a tag or label for performing functions such as purification, quantification, signal generation and the like. When the target biomolecule is known, the specific methods and means for detection and measurement thereof can be appropriately selected by those skilled in the art.

(Treatment and prevention)
In one aspect, the present disclosure provides treatment and / or prevention of a subject based on the identification of the subject's condition using the biomarkers of the present disclosure. The biomarkers of the present disclosure can identify the presence or absence of a disease in a subject, the state of the disease in the subject (malignancy, stage, etc.), the risk of the disease in the subject, the drug sensitivity of the subject, and the like. It may provide appropriate information for choosing methods for treating and / or preventing the body. In particular, the biomarkers of the present disclosure can identify subjects for whom the FGF19 / FGFR4 pathway targeting agent may be effective, and the present disclosure presents the treatment and / or prevention of such subjects with the FGF19 / FGFR4 pathway targeting agent. I will provide a. Here, the subject is of any disease that may be associated with the expression of FGF19, such as the proliferative diseases described herein, cholestasis, abnormal bile acid synthesis, chronic diarrhea due to poor bile acid malabsorption, metabolic syndrome, and the like. They may have non-alcoholic cholestasis, or insulin resistance, and may be diagnosed with these disorders. In a particularly preferred embodiment, the invention may provide FGF19 / FGFR4 pathway targeting agents (eg, lenvatinib) for use in patients determined to have high ST6GAL1 levels (eg, cancer patients).

The treatment and / or prevention of the present disclosure may be combined with any known treatment and / or prophylactic treatment or means (eg, anti-cancer treatment).

The treatment, prevention and / or diagnosis of the present disclosure can be performed in any subject. In one embodiment, the subject is a mammal such as a human, mouse, guinea pig, hamster, rat, rat, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee. Is. In a specific embodiment, the subject is a human.

(Pharmaceuticals, dosage forms, etc.)
The FGF19 / FGFR4 pathway targeting agents described herein and the biomarker detection agents of the present disclosure can be provided as various forms of compositions or pharmaceuticals. Also, in one embodiment, the disclosure is to treat a subject who has been shown to be highly malignant with a proliferative disorder by the methods described herein, or by the methods described herein. Provided are compositions comprising FGF19 / FGFR4 pathway targeting agents for treating subjects shown to be in the effective group. In one embodiment, the FGF19 / FGFR4 pathway target drug is an anticancer agent (eg, lenvatinib).

The routes of administration of the FGF19 / FGFR4 pathway targeting agents described herein (eg, lenvatinib, etc.) and the detection agents of the biomarkers of the present disclosure are effective in treating, preventing or detecting the diseases described herein. It is preferable to use one, for example, intravenously, subcutaneously, intramuscularly, intraperitoneally, orally. The administration form may be, for example, an injection, a capsule, a tablet, a granule, or the like. The aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. Further, the aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, NaOH or the like. Further, the therapeutic agent may contain, for example, a buffer (for example, a phosphate buffer), a stabilizer, or the like.

In general, the compositions, pharmaceuticals, detection agents, etc. of the present disclosure include therapeutically effective amounts of active ingredients or detectable amounts of detection means, as well as pharmaceutically acceptable carriers or excipients. As used herein, "pharmaceutically acceptable" is used in animals, and more specifically in humans, for use in government regulatory agencies, or in the pharmacopoeia or other generally accepted pharmacopoeia. It means that it is listed. As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle to which a therapeutic or detection agent is administered together. Such carriers can also be sterile liquids such as water and oil, including, but not limited to, petroleum, animal, plant or synthetic origins, sesame oil, soybean oil, minerals. Includes oil, sesame oil, etc. Water is the preferred carrier for oral administration of the drug. For intravenous administration of the pharmaceutical composition, saline and aqueous dextrose are preferred carriers. Preferably, aqueous saline solution, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, choke, silica gel, sodium stearate, glycerol monostearate, talc, chloride. Sodium, defatted milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene curing It contains 60 castor oil, sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like. The composition can also contain small amounts of wetting or emulsifying agents, or pH buffers, if desired. These compositions can also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. It is also possible to formulate the composition as a suppository using traditional binders and carriers such as triglycerides. Oral formulations can also include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable carriers are E. W. Described in Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such compositions contain a therapeutically effective amount of a therapeutic agent, preferably a purified form, along with an appropriate amount of carrier to provide the patient in a form that is appropriately administered. The formulation should be suitable for the mode of administration. In addition to these, for example, surfactants, excipients, colorants, fragrances, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity. It may contain an accelerator, a flavoring agent and the like.

In one embodiment of the present disclosure, "salt" includes, for example, an anionic salt formed of any acidic (eg, carboxyl) group, or a cationic salt formed of any basic (eg, amino) group. Salts include inorganic or organic salts and include, for example, the salts described in Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Further, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid and the like can be mentioned. In one embodiment of the present disclosure, a "solvate" is a compound formed by a solute and a solvent. For solvates, see, for example, J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953). If the solvent is water, the solvate formed is a hydrate. The solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.

In the present disclosure, various delivery systems can be used together when administering the drug, and such systems are used to administer the inhibitors and / or migratory agents of the present disclosure to appropriate sites. It is also possible to do. Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules: use of receptor-mediated endocytosis; construction of therapeutic nucleic acids as part of retroviral vectors or other vectors. be. Induction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. It is also possible and necessary to administer the drug by any of the preferred routes, eg by injection, by bolus injection, by absorption through the epithelium or mucocutaneous lining (eg, oral, rectal and intestinal mucosa, etc.). Inhalers or atomizers can be used, depending on the aerosolizing agent, and can also be administered with other bioactive agents. Administration can also be systemic or topical (eg, intratumor).

In a preferred embodiment, according to known methods, the composition can be formulated as a pharmaceutical composition adapted for administration to humans. Such compositions can be administered by injection. Typically, the composition for injection administration is a solution in a sterile isotonic aqueous buffer. If desired, the composition can also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dosage form and fed, for example in a sealed container such as an ampoule or sachet indicating the amount of activator, lyophilized powder or water-free concentrate. It can be supplied as a thing. If the composition is to be administered by infusion, it can also be dispensed using an infusion bottle containing sterile drug grade water or saline. If the composition is to be administered by injection, it is also possible to provide an ampoule of sterile water or saline for injection so that the ingredients can be mixed prior to administration.

The compositions, pharmaceuticals, therapeutic or prophylactic agents of the present disclosure can also be formulated in neutral or salt form or other prodrugs (eg, esters, etc.). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartrate, etc., isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, prokine. It includes those formed with a free amine group such as those derived from such as, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide and the like.

The amount of the composition, pharmaceutical, therapeutic or prophylactic agent of the present disclosure may vary depending on the nature of the disease, but one of ordinary skill in the art can be determined by standard clinical techniques based on the description herein. In addition, in vitro assays can optionally be used to help identify the optimal dosage range. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, within the range of any two values. There may be. The dosing interval is not particularly limited, but may be administered once or twice per 1, 7, 14, 21, or 28 days, for example, once or twice per range of any two values. May be good. The dose, administration interval, and administration method may be appropriately selected depending on the age and weight of the patient, symptoms, target organ, and the like. Further, the therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exerts a desired action. The effective dose can be estimated from the dose-response curve obtained in vitro or from an animal model test system.

The compositions, therapeutics, prophylactics or detectors of the present disclosure can be provided as kits.

In certain embodiments, the present disclosure provides drug packs or kits comprising one or more containers filled with one or more components of the compositions, therapeutics, prophylactics or detection agents of the present disclosure. Manufacture, use or sale for human administration by a government agency, as prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, optionally accompanying such containers. It is also possible to show information indicating the approval of.

The pharmaceutical formulation procedure of the therapeutic agent, preventive agent, etc. of the present disclosure is known in the art, and is described in, for example, the Japanese Pharmacopoeia, the United States Pharmacopeia, the Pharmacopoeia of other countries, and the like. Therefore, a person skilled in the art can determine an embodiment such as an amount to be used without conducting an excessive experiment, if the description of this specification is given.

(Animal model)
In one aspect, the present disclosure provides new animal models for cancer research (eg, mice, rats), methods of making them, or means for making them. In one embodiment, the present disclosure provides nucleic acids (eg, plasmids) containing oncogenes and unique barcodes. In one embodiment, the present disclosure provides a population of multiple types of nucleic acids, each containing multiple types of oncogenes and corresponding unique barcodes. By introducing such a population of nucleic acids into an animal, an animal into which various oncogenes have been randomly introduced can be produced. An animal into which various oncogenes are randomly introduced may show different responsiveness depending on the oncogene introduced into the treatment with a drug or the like, and thus the relationship between the oncogene and the treatment (for example, drug sensitivity). Is useful for studying. When using an animal into which a population of multiple types of nucleic acids, each containing multiple oncogenes and corresponding unique barcodes, has been introduced, use cDNA based on mRNA extracted from an animal-derived sample (eg, tumor tissue). Since the expression level of each oncogene can be estimated according to the number of readings of the unique bar code, the relationship between the quantitative information of each oncogene and the responsiveness of the animal to the treatment can be easily analyzed. be able to.

As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When "within a range" of "two values" is specified in the present specification, the range includes the two values themselves.

References such as scientific literature, patents, and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as they are specifically described.

The present disclosure has been described above with reference to preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments and examples specifically described in the present specification, and is limited only by the scope of claims.

If necessary, the handling of animals used in the following examples was carried out in accordance with the animal experiment guidelines of Shiga University of Medical Science. Specifically, the products described in the examples were used as the reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be used instead.

In the following examples, each abbreviation has the following meaning.
ANOVA: ANOVA AUC: Area under the curve CT: CT scan DMSO: Dimethyl sulfoxide ELISA: Enzyme-bound immunoadsorption assay FGF19: Fibroblast growth factor 19
FGFR: Fibroblast Growth Factor Receptor HCC: Hepatocellular Carcinoma IC50: 50% Inhibition Concentration KD: Knockdown OS: Overall Survival PFS: Progression-Free Survival PB: piggyBac
PDGFR: Platelet-derived growth factor receptor RFS: Recurrence-free survival ROC: Recipient behavior characteristics SIRAC: Stable isotope labeling using amino acids in cell culture medium TKI: Tyrosine kinase inhibitor VEGFR: Vascular endothelial growth factor receptor

(material and method)
Human serum and histological analysis Serum samples were treated with TKI at 76 HCC patients who underwent therapeutic hepatectomy at Osaka University and at other institutions participating in Osaka University and the Osaka Liver Forum. Obtained from 96 patients with advanced HCC. HCC tissue was obtained from 62 of the 76 HCC patients who were surgically resected. All patients provided written informed consent and the study design was consistent with the principles of the Declaration of Helsinki. The protocol for the study using patient serum and excised tissue was approved by the Institutional Review Board (IRB) Committee (IRB No. 19438) of Osaka University Hospital. Serum FGF19 and ST6GAL1 levels are enzymatically bound according to the manufacturer's protocol using an ELISA kit of human FGF19 (DF1900, R & D Systems, Minneapolis, Minnesota) and human ST6GAL1 (ab243669, Abcam, Cambridge, UK), respectively. It was examined by an immunoadsorption assay (ELISA). Tumor tissue was fixed with 10% neutral buffered formaldehyde solution and embedded in paraffin to make thin sections. Liver sections were stained with hematoxylin-eosin and primary FGF19 antibody (HPA036082, 1: 300, Sigma-Aldrich, St. Louis, Missouri). Using Citric Acid Recovery Buffer (Agilent) and Decloaking Chamber NxGen (Biocare Medical, Pacheco, CA), heat-mediated antigen recovery was performed to determine the FGF19 staining intensity of liver tumor sections using hybrid cell counting software (Keyence, Osaka). , Japan). Tumors with cytoplasmic FGF19 staining in more than 30% of tumor cells in two or more of the four high-power fields were classified into the FGF19 high group. The remaining tumors were classified in the FGF19 low group.

Statistical Analysis All data is displayed as a mean ± standard deviation (SD) in a bar graph or in a dot plot with a central horizontal bar showing the mean. Statistical analysis was performed using Student's t-test or Mann-Whitney U test to assess differences between unpaired groups of parametric or nonparametric distributions, respectively. One-way ANOVA and subsequent Tukey-Kramer post-test or Kruskal-Wallis test were performed for parametric or nonparametric multiple comparisons, respectively. Chi-square test or Fisher's exact test was used to analyze the category data. Correlation was evaluated using Pearson's product moment correlation coefficient. Differences in OS, progression-free survival (PFS), and recurrence-free survival (RFS) were analyzed using the Kaplan-Meier method and the logrank test. A univariate Cox proportional hazards regression model was used to analyze factors associated with OS improvement. To evaluate the diagnostic performance of serum biomarkers, receiver operating characteristic (ROC) curve analysis was performed and predictive detection power was evaluated using the area under the curve (AUC). The cutoff value was determined by J statistics of Youden (Cancer 1950; 3: 32-35). Otherwise, the statistical analysis used is shown in the legend in the figure. A p-value <0.05 was considered to indicate statistical significance. Prism ver.8.4.2 for Windows (GraphPad Software, San Diego, CA) and JMP® 13 (SAS Institute Inc., Cary, NC) were used for the analysis.

Cell Line Human Liver Cancer Cell Line Hep3B was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The Huh-7 human liver cancer cell line was obtained from the Japanese Cancer Research Resource Bank (JCRB, Ibaraki, Japan). These cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) or RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. It was confirmed that all cells were free of pathogens and mycoplasma.

Mass spectrometry of cell lines 10% dialysate FBS and heavy isotopes 13C6-L-lysine-2HCl and 13C6-L-arginine according to standard isotope labeling (SILAC) protocol with amino acids in cell culture. Subcultured in DMEM containing -2HCl or the light isotopes L-lysine-2HCl and L-arginine-2HCl (Nat Protoc 2006; 1: 2650-60). According to the manufacturer's instructions, cells were transfected with siRNA at a final concentration of 10 nM using RNAiMAX. The following siRNAs were used: siControl (Thermo Fisher Scientific, 4390844) and siFGF19 (Thermo Fisher Scientific, s19354). After 48 hours of siRNA transfection, the medium was replaced with fresh medium containing 0.1% FBS. Conditional medium was collected 48 hours after incubation, centrifuged at 1,500 rpm to remove cell debris, filtered through a 0.22 μm filter (Promega, Madison, WI), and Amicon Ultra-15 (nominal fractional molecular weight (nominal fraction molecular weight) (nominal fraction molecular weight). NMWL): 3,000, Merck, Hessen, Germany) concentrated about 10-fold. To obtain a whole cell extract, 2x10 ⁷ cells were added with cOmplete Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland) to 1% sodium dodecyl sulfate (SDS) and 50 mM ammonium bicarbonate (ABC). ) Was dissolved in 1 ml of PBS and boiled at 95 ° C. for 5 minutes. Protein concentrations in conditioned medium and whole cell extract were determined by detergent-compatible (DC) protein assay (Bio-Rad Laboratories, Berkeley, California).

Equal amounts of light isotope-labeled and heavy isotope-labeled samples were mixed and precipitated with methanol-chloroform. The sample was then redissolved in 8M urea and sonicated for 15 minutes. 150 micrograms of each sample were diluted 4-fold with 50 mM ABC and digested with trypsin (trypsin amount: protein weight, 1:50) at 37 ° C. for 18 hours. The tryptic digested peptide was acidified with 1% trifluoroacetic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and desalted using an Oasis® HLB column (Waters, Milford, MA).

The peptide (100 μg) was fractionated by high pH reverse phase liquid chromatography (RPLC) using an LC-20AB device (Shimadzu, Kyoto, Japan). Peptide separation was performed on an L-volume 3 C18 analytical column (CERI) containing 5 μm particles at a flow rate of 0.2 μml / min. The solvent was prepared with 2% acetonitrile and 5 mM ABC (pH 10) as mobile phase A and 90% acetonitrile and 5 mM ABC (pH 10) as mobile phase B. Separation was performed on the following linear gradient: 1% B to 7% B in 1 minute, 7% B to 27% B in 22 minutes, 27% B to 45% B in 6 minutes, 45% B in 1 minute. From 95% B. The samples were divided into 34 fractions and finally grouped into 15 pools so that they were discontinuous.

Pools were analyzed by LC-MS / MS using a Q Exactive mass spectrometer (Thermo Fisher Scientific) combined with the UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific). A nano HPLC capillary column (inner diameter 150 mm × 75 μm; Nikkyo Technos, Tokyo, Japan) was used as an analytical column equipped with a nanoelectrospray ion source. RPLC was performed with a linear gradient of acetonitrile from 5% to 40% in 0.1% formic acid at a flow rate of 300 ln / min. Full scan spectra of the MS1 survey were acquired in the range of 400-1600 m / z. Twenty strongest ions per MS scan were selected for fragmentation due to high energy collision dissociation within the collision cell.

The acquired mass spectrometric data was processed using MaxQuant version 1.5.7.0 supported by the Andromeda search engine (Nat Biotechnol 2008; 26: 1367-72). Tandem mass spectra were searched against a database of human proteins (released April 2019) obtained from UniProt combined with 262 common impurities. To consider the uptake of heavy isotopes, a fixed modification of 6.012129 additional mass units for lysine and arginine residues was used in the database search. For peptide identification, the search criteria were set as follows: trypsin specificity, tolerance for C-terminal cleavage of arginine or lysine at proline binding, tolerance for up to 2 cleavage errors, cysteine residue as a fixed modification. Carbamide methylation, and methionine oxidation as a variable modification. The false positive rate for protein and peptide identification was less than 0.01. Peptides identified in the reverse database and the potential impurity database were excluded.

siRNA transfection siRNA (s19353, s19354, or s19355, Thermo Fisher Scientific) or negative control siRNA (4404020, Thermo Fisher Scientific) with Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) as previously described. FGF19 was knocked down in HCC cells (Suemura et al., CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma. Cancers (Basel) 2019; 11).

Real-time PCR analysis Total RNA was isolated from tissue or cells and reverse transcribed into cDNA as previously described (Cell Death Differ 2019; 26: 470-86). The expression of mRNA was measured by quantitative real-time reverse transcription PCR (RT-qPCR) using THUNDERBIRD RT-qPCR master mix (Toyobo, Osaka, Japan) and TaqMan probe (Thermo Fisher Scientific). Target gene expression is normalized to beta-actin expression and presented as a multiple increase compared to control group levels.

(result)
ST6GAL1 is a tumor-derived secreted protein positively regulated by FGF19 in HCC Next, tumor-derived secreted proteins were examined to identify serum biomarkers of HCC promoted by FGF19. To this end, FGF19-expressing Hep3B and Huh7 HCC cells with and without FGF19 knockdown were used to perform stable isotope-labeled (SILAC) -based cell proteome and secretomic analysis with amino acids in cell culture medium. (Fig. 1).

As a result, the proteins whose expression was decreased or increased (more than 2-fold) by FGF19 knockdown are shown below. In addition, the protein not detected in the FGF19 knockdown sample and detected in the corresponding non-knockdown sample is regarded as a decrease, and the protein detected in the FGF19 knockdown sample and not detected in the corresponding non-knockdown sample is increased. I considered it.

The following proteins were commonly downregulated by FGF19 knockdown in secretome analysis of both Hep3B and Huh7 cells.
A1BG, A2M, ACSL4, AGRN, AHSG, APPL2, APOA2, APOC1, APP, ARID1B, ATP6AP2, B3GAT2, CCL15, CCL20, CLSTN3, CLU, CP, CTGF, DNAJB11, EXTL2, F2, F5 FURIN, GBA, GCNT2, GDF15, GPR126, HNRNPA3, ICAM1, LDLR, LGALS3BP, LRG1, LYZ, MAN1A1, MEP1A, NTS, ORM1, PCOLCE2, PPT1, PROS1, SDC4, SDCBP SPTBN4, ST6GAL1, STC2, TFPI, TFRC, TNFRSF11B, TOR1B, TTR, UXS1, VWA1

The following proteins were commonly downregulated by FGF19 knockdown in whole proteome analysis of both Hep3B and Huh7 cells.
ANLN, ANXA1, ARMX1, ATP1A4, CCL20, CTGF, DYNLL2, FIBP, FYTTD1, GDF15, H2AFX, HMCES, HSPA13, IFRD1, INPP1, IREB2, ITGA2, MAP2K3, NT5E, FORM1 SIL1, SLC7A1, SLC7A11, SMG6, ST6GAL1, SUGP2, THBS4, TMPO, TUBE1, UBE2B, VAMP3 (VAMP2), YIPF4

The following proteins were commonly expressed in all four samples by FGF19 knockdown.
ACTG1, ALDH9A1, ANPEP, CDH6, COL18A1, DCD, FLNC, FSCN1, GSTM2, GSTM3, HIST1H4A, HIST2H3A, HPX, HSPB1, IL1RAP, S100A7, TLN1, YWHAH

CTGF, CCL20, GDF15, ST6GAL1, ORM1, and SERPINI1 were identified as proteins whose expression was reduced by FGF19 knockdown in all four samples. These expressions were reduced in both lysates and supernatants of the two cell lines when FGF19 was suppressed by siRNA (FIG. 2). Next, when the expression levels of each of the six genes when FGF19 was inhibited by siRNA were evaluated, it was confirmed that CTGF, GDF15, and ST6GAL1 were significantly down-regulated by FGF19 knockdown by two types of siRNA. (Fig. 3). The correlation between gene expression of FGF19 and gene expression of two secreted proteins in HCC cell lines and human HCC tissues was further evaluated. FGF19 levels were significantly correlated with ST6GAL1 levels, but not with GDF15 levels in 9 human liver cancer cell lines (FIG. 4) and HCC tissues from the TCGA cohort (FIG. 5). In summary, it was shown that the secreted protein ST6GAL1 is the protein most positively correlated with FGF19 in HCC. Therefore, ST6GAL1 can be an alternative serum marker for FGF19-promoting HCC.

Serum ST6GAL1 is a biomarker of FGF19-promoting high-grade HCC Next, we investigated the association between serum ST6GAL1 levels and tumor FGF19 expression in 62 HCC patients who underwent therapeutic surgical resection.

Immunohistochemical analysis showed that about a quarter of HCC patients showed high levels of FGF19 expression in tumor cells. Patients with FGF19 high HCC showed significantly advanced disease and short RFS after therapeutic resection (Fig. 5), suggesting that FGF19 high HCC is very malignant, a previous report (Dig Dis Sci 2013). It is consistent with 58: 1916-1922). Serum ST6GAL1 levels were significantly higher in FGF19 high HCC patients than in FGF19 low HCC patients (FIG. 6). In addition, using the 19.1 ng / ml cutoff serum ST6GAL1 value determined by the Youden index, patients with FGF19 high HCC were identified with high sensitivity and specificity (FIG. 7). In addition, serum ST6GAL1 levels were positively correlated with tumor size and stage (FIG. 8), with patients with high serum ST6GAL1 levels having significantly shorter RFS than those with low levels (FIG. 9). In contrast, serum FGF19 levels at the tumor site were not different between FGF19 high HCC patients and FGF19 low HCC patients and could not be used to distinguish groups (FIG. 10). In addition, serum FGF19 levels did not correlate with stage or RFS in HCC patients (FIG. 11). From the above results, it was clarified that ST6GAL1 is a highly reliable serum biomarker for identifying FGF19-expressing HCC patients showing high malignancy.

Serum ST6GAL1 is a biomarker of lenvatinib-sensitive HCC Based on experimental evidence that FGF19-promoting HCC exhibits high lenvatinib susceptibility, baseline serum ST6GAL1 levels are also assumed to be useful biomarkers for predicting lenvatinib susceptibility in HCC patients. bottom. To test this hypothesis, pretreatment serum ST6GAL1 levels were examined in 96 advanced HCC patients who were later treated with lenvatinib or sorafenib and analyzed for prognosis. There was no difference in serum ST6GAL1 levels (FIG. 12) or OS (FIG. 13A) between lenvatinib-treated and sorafenib-treated patients. Patients were then divided into ST6GAL1 high and ST6GAL1 low groups based on the cutoff serum ST6GAL1 levels used to identify FGF19 high HCC. In 62 HCC patients with low serum ST6GAL1 levels, OS was not different between lenvatinib-treated and sorafenib-treated patients (FIG. 13B). On the other hand, among 34 HCC patients with high serum ST6GAL1 levels, lenvatinib-treated patients showed significantly longer OS than sorafenib-treated patients (FIG. 13C). In addition to Child-Pugh score, Barcelona clinical liver cancer (BCLC) stage, and serum alphafet protein (AFP) levels, lenvatinib treatment treatment also predicts OS improvement in the high ST6GAL1 group by univariate Cox proportional hazard analysis. It was identified as one of the factors (Table 7).

These results suggest that clinical observations may help ST6GAL1 select HCC patients whose lenvatinib therapy offers superior survival benefits over sorafenib therapy. It is expected that lenvatinib is effective for subjects stratified based on the expression of ST6GAL1 mainly due to the FGFR4 inhibitory activity of lenvatinib.

The present disclosure provides biomarkers that correlate with FGF19 expression. Based on this finding, a new method for evaluating a condition such as cancer (for example, drug susceptibility) without imposing an excessive burden on the patient is provided.

Claims (15)

A method in which the expression of a biomarker selected from the biomarker of group (A) and the biomarker of group (B) in a subject having a proliferative disease is used as an index of the malignancy of the proliferative disease.
Including the step of measuring the expression of the biomarker in the sample derived from the subject.
The expression level of the biomarker selected from the measured biomarkers in the group (A) exceeds the reference value, or the expression level of the biomarker selected from the measured biomarkers in the group (B) exceeds the reference value. Below, it indicates that the proliferative disorder in the subject is likely to be highly malignant.
Method. A method in which the expression of a biomarker selected from the biomarker of group (A) and the biomarker of group (B) in a subject having a disease is used as an index of the efficacy of a FGF19 / FGFR4 pathway target drug.
Including the step of measuring the expression of the biomarker in the sample derived from the subject.
The expression level of the biomarker selected from the measured biomarkers in the group (A) exceeds the reference value, or the expression level of the biomarker selected from the measured biomarkers in the group (B) exceeds the reference value. Below, the subject is indicated to be an effective group against the FGF19 / FGFR4 pathway targeting agent.
Method. The method of claim 1 or 2, wherein the sample comprises a blood sample. The method according to any one of claims 1 to 3, wherein the biomarker is selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1 and SERPINI1. The method according to any one of claims 1 to 3, wherein the biomarker is ST6GAL1. The method according to any one of claims 1 to 5, wherein the disease is cancer. The method of claim 6, wherein the cancer comprises liver cancer, breast cancer, pancreatic cancer or colorectal cancer. The method according to any one of claims 2 or 3 to 7, wherein the FGF19 / FGFR4 pathway target drug is an anticancer agent. 12. The one according to claim 2, wherein the FGF19 / FGFR4 pathway target drug is selected from the group consisting of lenvatinib, physogatinib, FGF401, H3B-6527 and BLU9931, claim 2 or dependent on claim 2. Method. 9. The method of claim 9, wherein the FGF19 / FGFR4 pathway target drug comprises lenvatinib. A method for testing the expression of FGF19 in a subject, comprising the step of measuring the expression of a biomarker selected from the biomarker of group (A) and the biomarker of group (B) in a sample derived from the subject. 11. The method of claim 11, wherein a disease that abnormally expresses FGF19 is indicated based on the measured expression of the biomarker. 12. The method of claim 11 or 12, wherein the biomarker is selected from the group consisting of CTGF, CCL20, GDF15, ST6GAL1, ORM1 and SERPINI1. The method according to any one of claims 11 to 13, wherein the biomarker is ST6GAL1. A kit for use in the method according to any one of claims 1 to 14, comprising the biomarker detection agent.

Images