KR20220132280A - Anti-bromodomain-containing protein 2-specific autoantibody cancer biomarker, antigen composition for detection thereof, and liver cancer diagnosis method using the same - Google Patents

Abstract

The present invention relates to an autoimmune antibody (autoantibody) cancer biomarker that specifically binds to bromodomain-containing protein 2 (BRD2), to a composition for diagnosing liver cancer including a polypeptide containing an epitope for detecting the autoimmune antibody, and to a method for diagnosing liver cancer using the same. A composition for diagnosing liver cancer including a polypeptide containing an epitope for detecting BRD2-specific reactive autoimmune antibody (autoantibody) and a method for diagnosing liver cancer using the same according to the present invention can diagnose liver cancer by using non-invasive biological samples such as blood, plasma, serum, and lymph without requiring a biopsy. Also, the method is useful for diagnosing liver cancer because the autoimmune antibody detection sensitivity and specificity are remarkably high.

Description

BRD2-specific autoimmune antibody cancer biomarker, antigen composition for detection thereof, and liver cancer diagnosis method using the same

The present invention relates to an autoimmune antibody (autoantibody) cancer biomarker that specifically binds to Bromodomain-containing protein 2 (BRD2), a polypeptide comprising an epitope for detection of the anti-BRD2 autoimmune antibody It relates to a composition for diagnosing liver cancer and a method for diagnosing liver cancer using the same.

Recently, with the great development of the biomedical field, in addition to the existing chemotherapy, antibody therapy, immunotherapy, etc. are used as anticancer therapies, and cancer is no longer causing death, but is changing into a treatable disease. However, it is still difficult to treat cancer that has developed, and the cost burden is high. Accordingly, the need for prevention and early diagnosis of cancer to prevent the onset of cancer in advance is further emerging.

Cancer diagnostic methods are mainly used to determine whether or not cancer occurs. Existing cancer diagnostic methods include endoscopy, imaging tests, and nuclear medicine tests. In vitro diagnostic methods for tumor markers that detect tumor biomarkers established through molecular biological and biochemical studies are being used. In addition, companion diagnosis to confirm the suitability and adaptability of drug administration for effective implementation of various recently attempted anticancer therapies is also being studied as an important field. The field is expanding with the concept of liquid biopsy.

Effective in vitro diagnostic tumor markers are useful in diagnosing cancer because they can confirm cancer with a simple blood test and determine the degree of progression. Most of the in vitro diagnostic tumor markers currently used in clinical practice are antigens specifically expressed and secreted by cancer cells. Alpha-fetoprotein (AFP), Cancer antigen 125 (CA125), carcinoembryonic antigen (CEA), and prostate-specific antigen ), and their blood levels are measured by ELISA (Enzyme-Linked Immunosorbent Assay). However, due to the low sensitivity and specificity of the existing methods for cancer diagnosis, they are currently used only as an auxiliary means for cancer diagnosis, and more effective tumor markers are discovered and put into practice.

The large-scale analysis of blood biomarkers presented various examples in which proteins, nucleic acids, circulating tumor cells, etc. leaked from cancer tissues can be used as tumor markers. In addition, as it has been reported that cancer tissue-derived exosomes secreted into the blood contain a variety of cancer-derived substances such as DNA, RNA, and proteins, many attempts to diagnose cancer by analyzing exosomes are underway. However, since the amount of cancer cell-derived antigen and exosomes is related to the size of cancer tissue, it is difficult to detect a small amount of exosomes in the early stage of cancer development. Diagnostics are being tried.

Various protein antigens contained in cancer cell-derived exosomes can be used as markers by themselves, but autoimmune antibodies (autoantibodies) induced by them are also being studied as biomarkers. Antigens contained in exosomes are small enough to be detected by in vitro diagnostic methods, but are judged to be at a level capable of inducing antibodies by stimulating the body's immune system. Autoimmune antibodies induced with a small amount of antigen have an advantage as an in vitro diagnostic marker because their expression level is amplified during the antibody induction process and has a very stable form for in vitro analysis due to the nature of the protein. In addition, it is easy to use as a biomarker for early diagnosis of cancer because numerous existing analysis systems established for antibody detection can be used for autoimmune antibody detection.

In fact, anti-p53 autoimmune antibodies identified in the blood 2 to 3 years before the onset of lung cancer are being studied as important early detection markers for cancer (Yen-Heng Lin, et al., Detection of anti-p53 autoantibodies in saliva using microfluidic chips). For the rapid screening of oral cancer, RSC Adv., 2018, 8, 15513-15521), research to discover other autoimmune antibodies capable of similar functions as biomarkers has been ongoing for the past 10 years. However, when a recombinant protein or a linear peptide is used to detect a cancer-related autoimmune antibody, most cases have low autoimmune antibody detection sensitivity, so examples of practical use as a cancer diagnosis method are limited.

Under this background, the present inventors studied human autoimmune antibody cancer biomarkers using cancer model mice ( FIG. 1 ). As a result, autoimmune antibodies exhibiting a specific response to Bromodomain-containing protein 2 (BRD2) as cancer biomarkers. The present invention was completed by deriving a polypeptide containing an epitope for the detection of the anti-BRD2 autoimmune antibody and constructing an autoantibody biomarker detection method that improves the autoimmune antibody detection sensitivity. .

One object of the present invention is to provide a fragment comprising an autoimmune antibody or complementary determining regions (CDR) thereof, which specifically binds to Bromodomain-containing protein 2 (BRD2).

Another object of the present invention is to provide a hybridoma cell line that produces the autoimmune antibody.

Another object of the present invention is to specifically bind to the autoimmune antibody, and to prepare an epitope polypeptide comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10. will provide

Another object of the present invention is to provide a composition for diagnosing liver cancer, comprising an agent for measuring the expression level of the autoimmune antibody or a fragment comprising a complementarity determining region thereof.

Another object of the present invention is to provide a kit for diagnosing liver cancer comprising the composition.

Another object of the present invention is to provide a method of providing information for diagnosing liver cancer, comprising detecting a fragment comprising an autoimmune antibody or its complementarity determining region that specifically binds to BRD2 using a composition. will do

Another object of the present invention is to (i) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 from an individual; (ii) administering a candidate substance for the treatment of liver cancer to the subject; (iii) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 from the subject to which the candidate substance has been administered; and (iv) when the expression level measured in step (iii) is reduced compared to the expression level measured in step (i), determining the candidate substance as a liver cancer treatment agent. is to provide

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

As one aspect for achieving the above object, the present invention provides a fragment comprising an autoimmune antibody or complementary determining regions (CDR) thereof that specifically binds to Bromodomain-containing protein 2 (BRD2). do.

As used herein, the term "Brmodomain-containing protein 2 (BRD2)" refers to a part of a family of bromodomain and extra-terminal motif (BET) proteins in mammals, including spermatogenesis and It is known to be involved in the process of folliculogenesis and to regulate gene transcription through remodeling of hyperacetylated chromatin. The BRD2 gene encoding BRD2 is mapped to the major histocompatability complex (MHC) class II region of chromosome 6p21.3, but is not expected to be involved in the immune response according to sequence comparison results. In addition, the gene is related to juvenile myoclonic epilepsy, a general form of epilepsy that occurs in adolescence, and is known to have low tissue expression specificity.

The specific nucleotide sequence and protein information of the BRD2 and the BRD2 gene encoding it are known from NCBI (NCBI Reference Sequence: NG_042801.1).

BRD2 of the present invention has not been reported as a cancer prognostic marker to date, nor is it known about the generation of anti-BRD2 autoimmune antibodies. Accordingly, the present invention is meaningful in that the relationship between liver cancer and BRD2, an anti-BRD2 autoimmune antibody, and a polypeptide mimetic composed of an amino acid sequence similar to its BRD2 epitope were identified for the first time, and used for the diagnosis of liver cancer.

As used herein, the term "autoimmune antibody (autoantibody)" refers to an antibody that specifically reacts with its own body composition, and is also referred to as an autoantibody. In general, an individual does not produce an antibody because it does not provoke an immune response to the substance it possesses. However, in a special case, an antibody is also generated against the original substance, and the generated antibody is called an “autoimmune antibody”. The case where the autoimmune antibody is generated may include when a specific disease occurs, for example, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, or cancer. Autoimmune antibodies may be produced when the disease develops.

For the purposes of the present invention, the autoimmune antibody may be an antibody generated against a tumor-associated antigen (TAA) involved in abnormal cancer cell growth.

In one embodiment of the present invention, the expression of the XC246 antigen ( FIGS. 2 to 3 ) in Hepa1c1c7, a mouse liver cancer cell, and various human cancer cells is confirmed, and the isotype of the anti-XC246 antibody binding thereto is the IgM type. was done (FIGS. 4 to 5). As a result of analyzing the nucleotide sequence of the IgM-type anti-XC246 autoimmune antibody, a heavy chain protein comprising the amino acid sequence of SEQ ID NO: 11 (FIG. 23) and a light chain protein comprising the amino acid sequence of SEQ ID NO: 12 (FIG. 24) were obtained. It was confirmed that it is an autoimmune antibody containing

Generally, one antibody molecule has two heavy chain proteins and two light chain proteins, and each heavy chain and light chain includes a mutation region at its N-terminus. Each variant region consists of three complementarity determining regions (CDRs) and four framework regions (FRs), which determine the antigen-binding specificity of the antibody and form the structure of the variant region. It exists as a relatively short polypeptide sequence maintained in regions. The heavy and light chains can be used individually or together depending on the purpose, and a number of CDR sequences and a free combination of light and heavy chains are possible according to conventional genetic engineering methods as desired by those skilled in the art. Furthermore, it is apparent that the nucleic acid sequence encoding the sequence is also included in the present invention.

The anti-XC246 autoimmune antibody of the present invention comprises a polynucleotide encoding a heavy chain comprising two full lengths or an immunologically active fragment of an antibody molecule capable of achieving antibody-antigen binding. Anti-XC246 antibodies of the present invention also include polynucleotides encoding two full-length light chains or fragments with immunological activity of an antibody molecule capable of achieving antibody-antigen binding. A fragment having immunological activity of an antibody molecule refers to a fragment having an antigen-binding function, for example, (i) a light chain variable region (VL), a heavy chain variable region (VH), and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a monoclonal antibody; (iv) a dAb fragment consisting of the VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv) joined by a peptide linker that joins the VH domain and the VL domain to form an antigen binding site; (viii) a bispecific single-chain Fv dimer (PCT/US92/09965) and (ix) a multivalent or multispecific fragment produced by gene fusion, diabody WO94/13804), etc. , but is not limited thereto.

In one embodiment of the present invention, in order to identify an anti-XC246 antibody-specific antigen protein, the antigen protein is identified as an anti-XC246 antibody ( FIGS. 6 to 7 and 8 ), and after immunoprecipitation, it is analyzed by mass spectrometry. (FIGS. 9 to 10), as a result of confirming whether the anti-XC246 antibody response of the selected proteins was reduced, it was confirmed that the anti-XC246 antibody-specific antigen protein was BRD2 (FIGS. 11 to 13). Accordingly, hereinafter, the anti-XC246 antibody is an anti-XC246 autoimmune antibody, an autoimmune antibody that specifically binds to XC246, an anti-BRD2 antibody, an anti-BRD2 autoimmune antibody, an autoimmune antibody that specifically binds to BRD2, etc. can be mixed.

In one embodiment of the present invention, in order to identify the epitope binding to the anti-BRD2 autoimmune antibody, 7 amino acids are expressed in a random sequence, and a total of 9 amino acids including cysteine residues at both ends form a cyclic structure. A phage library expressing a cyclic peptide was used (FIG. 14), and specifically, phages that specifically bind to the anti-BRD2 autoimmune antibody were selected (FIGS. 15 to 18), while the selected phage and anti-BRD2 By confirming the reactivity with the autoimmune antibody, the antigenic determinant as described above was determined.

In the present invention, the anti-BRD2 autoimmune antibody is C SSMFLPS C shown in SEQ ID NO: 1 ( C : cysteines at both ends of a 9 amino acid sequence form a disulfide bond using a sulfonyl residue of each amino acid) C. cysteine residue participating in disulfide bond is denoted by C ), C SSQWLPF C set forth in SEQ ID NO: 2, C TSALFPW C set forth in SEQ ID NO: 3, C VSASFPF C set forth in SEQ ID NO: 4, SEQ ID NO: 5 C NQVAYPW C set forth in SEQ ID NO: 6, C FSALYPW C set forth in SEQ ID NO: 6, C CRLRWPH C set forth in SEQ ID NO: 7, C TSSFFPH C set forth in SEQ ID NO: 8, C TSVFLPH C set forth in SEQ ID NO: 9 and SEQ ID NO: 10 It may be one that recognizes and binds a polypeptide consisting of any one or more amino acid sequences selected from the group consisting of the described C STAMALV C as an epitope determinant, that is, an epitope sequence. The amino acids are arginine (Arg, R), lysine (Lys, K), histidine (His, H), glutamic acid (Glu, E), aspartic acid (Asp, D), glycine (Gly, G), alanine, respectively (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), phenylalanine (Phe, F), tryptophan (Trp, W), proline ( Pro, P), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gln, Q).

Specifically, the anti-BRD2 autoimmune antibody may be an anti-BRD2 autoimmune antibody that recognizes and binds the amino acid sequence of SEQ ID NO: 9.

Another aspect of the present invention provides a hybridoma cell line producing the autoimmune antibody of the present invention.

The terms used herein are the same as described above.

As used herein, the term "hybridoma" refers to a cell created by artificially fusion of two different types of cells, using a substance that causes cell fusion, such as polyethylene glycol, or a virus of a certain species. Cells are fused cells. A hybridoma integrates different functions of different cells into one cell, and a representative example of a hybridoma is a lymphocyte. In particular, hybrid cells obtained by fusion of myeloma cells and B cells, which are precursors of antibody-producing cells, among lymphocytes in the spleen or lymph nodes, are widely applied in research and clinical practice because they produce monoclonal antibodies. In addition, T cells that produce lymphokines (bioactive substances) and hybridomas, which are tumor cells, are also being put to practical use. The hybridoma producing the autoimmune antibody of the present invention can be used by appropriately modifying cells known in the art by those skilled in the art.

In an embodiment of the present invention, by fusion of Sp2/0 and B cells, which are mouse myeloma cells, After incubation, only the B cell hybridomas for which the cancer cell reactive antibody was confirmed were selected and the anti-BRD2 autoimmune antibody of the present invention was isolated therefrom. The B cell hybridoma cell line producing the anti-BRD2 autoimmune antibody was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on November 23, 2020, and was given an accession number KCTC 14386BP. Specifically, the hybridoma cell line may be a cell line with accession number KCTC 14386BP.

Another aspect of the present invention binds specifically to the autoimmune antibody of the present invention, and consists of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10, epitope) Polypeptides are provided.

The terms used herein are the same as described above.

The antigenic determinant polypeptide is prepared in the form of CX ₇ C each additionally containing cysteines (Cys; C) at both ends, so that an antibody-specific reaction is possible when a stable cyclic structure is formed. Such a polypeptide can be used as an epitope determinant polypeptide mimetic for detecting the anti-BRD2 autoimmune antibody of the present invention. SEQ ID NOs: 1 to 10 of the present invention may be the sequence of X ₇ .

In one embodiment of the present invention, in order to identify the epitope binding to the anti-BRD2 autoimmune antibody, 7 amino acids are expressed in a random sequence, and a total of 9 amino acids including cysteine residues at both ends form a cyclic structure. A cyclic peptide expression phage library to form was used, as described above.

Another aspect of the present invention provides a composition for diagnosing liver cancer, comprising an agent for measuring the expression level of an autoimmune antibody of the present invention or a fragment comprising a complementarity determining region thereof.

The terms used herein are the same as described above.

As used herein, the term "liver cancer" refers to a weak tumor originating from hepatocytes, which occupy most of the liver, and is caused by metastasis of all types of malignant tumors (eg, intrahepatic cholangiocarcinoma) or cancers of other organs to the liver. including metastatic liver cancer. The prognosis of liver cancer varies depending on the number and size of tumors, vascular invasion, etc. The main cause of death in liver cancer patients is not liver cancer itself, but deterioration of liver function (liver failure) due to liver cancer progression.

The existing method of detecting the overexpressed protein itself in cancer has a problem in that it is not suitable for use as a cancer diagnostic marker because the half-life is reduced when the cancer-specific proteins are secreted into the blood, and thus detection is not easy. However, the anti-BRD2 autoimmune antibody of the present invention has a long half-life, high detection sensitivity, and can be easily non-invasively collected from a patient, such as blood, and thus can be easily used as a cancer diagnostic marker.

As used herein, the term “diagnosis” refers to ascertaining the presence or characteristics of a pathological condition. For the purpose of the present invention, it may include not only determining whether liver cancer occurs, but also determining the course of the subject after anticancer treatment, such as recurrence, metastasis, drug reactivity, resistance, and the like.

As used herein, the term "agent for measuring the expression level of an autoimmune antibody or a fragment comprising a complementarity determining region" refers to a marker whose expression is increased in whole blood, serum and plasma, lymphatic and intercellular fluid of an individual suffering from or suspected of liver cancer. It means a molecule that can be used for detection of a marker by checking the expression level of the anti-BRD2 autoimmune antibody, specifically, a polypeptide that specifically binds to the autoimmune antibody or a fragment containing a complementarity determining region thereof. have.

The polypeptide may include an antigenic determinant comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10 that recognize and bind the autoimmune antibody or a fragment containing a complementarity determining region thereof. have. The polypeptide comprising the antigenic determinant region may be prepared to have a stable cyclic structure by additionally including cysteines at both ends, as described above.

In addition, the polypeptide comprising the epitope may be prepared in the form of a carrier protein and a fusion protein in order to secure an effective expression body of the epitope.

As used herein, the term "carrier protein" refers to a protein or a fragment of a protein that binds to a target protein or polypeptide and effectively maintains the expression of an antigenic determinant, for example, GFP (green fluorescence protein), HSA (human serum albumin), maltose binding protein (MBP), streptavidin, and the like, but any carrier protein known in the art may be used without particular limitation.

Another aspect of the present invention provides a kit for diagnosing liver cancer comprising the composition of the present invention.

The terms used herein are the same as described above.

The kit of the present invention may include a composition for diagnosing liver cancer including an agent for measuring the expression level of an anti-BRD2 autoimmune antibody or a fragment including a complementarity determining region thereof.

The antigen that specifically binds to the anti-BRD2 autoimmune antibody or fragment containing the complementarity determining region thereof includes all proteins capable of specifically binding to the autoimmune antibody or the fragment containing the complementarity determining region thereof. , but not limited to a specific protein or polypeptide. Specifically, as long as the antigen can be recognized by the anti-BRD2 autoimmune antibody or the fragment including the complementarity determining region thereof, it may include all fragments or variants thereof. Such an antigen may consist of 1 to 2000 amino acids, and may consist of 5 to 16 amino acids as a specific example for the purpose of the present invention, but is not limited thereto. More specifically, the antigen may be a protein comprising an epitope having a structure that can be recognized by the anti-BRD2 autoimmune antibody marker of the present invention. The size or type of the sequence is not particularly limited as long as the epitope has a structure recognizable by the anti-BRD2 autoimmune antibody of the present invention. It may be expressed as a peptide, and as another example, it may be expressed as a polypeptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 10, as described above.

The kit of the present invention provides primers, probes, or selectively for measuring the expression level of an autoimmune antibody that specifically binds to BRD2, a marker for diagnosing liver cancer, or a fragment comprising a complementarity determining region thereof, as well as an antibody recognizing the marker. One or more other component compositions, solutions or devices suitable for analytical methods may be included.

In addition, the kit may use immunological detection of the antibody to measure the expression level of an autoimmune antibody that specifically binds to BRD2 or a fragment comprising a complementarity determining region thereof. The immunological detection may be achieved through an antibody-antigen reaction between the anti-BRD2 autoimmune antibody of the present invention or a fragment comprising a complementarity determining region thereof, and an antigen specifically binding thereto.

As used herein, the term "antigen-antibody complex" refers to a combination of an autoimmune antibody, a liver cancer marker, or a fragment containing a complementarity determining region thereof, and an antigen specific thereto, and the amount of the antigen-antibody complex formed is determined by the detection label ( It can be quantitatively measured through the magnitude of the signal of the detection label). In the present invention, the antigen-antibody complex may be an anti-BRD2 autoimmune antibody or a fragment including a complementarity determining region thereof, and an antigenic determinant or antigen protein binding product that reacts specifically thereto.

The detection label may be selected from the group consisting of a chromogenic enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, a radioisotope, a substrate, and a chromogenic substrate solution, but is not necessarily limited thereto.

When an enzyme is used as the detection label, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine. Esterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphophenolpyruvate decarboxylase, β-latamase, and the like, but are not limited thereto. The fluorescent material includes, but is not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrine, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. The ligand may include, but is not limited to, a biotin derivative. The light-emitting material includes, but is not limited to, acridinium ester, luciferin, luciferase, and the like. The microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecule include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W(CN) ₈ , [Os(bpy) ₃ ] ²⁺ , RU(bpy) ₃ ] ²⁺ , [MO(CN) ₈ ] ^4- , and the like, but are not limited thereto. The radioactive isotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like, but are limited thereto. doesn't happen The substrate includes, but is not limited to, a nitrocellulose membrane, a 96-well plate synthesized from a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, and a glass slide glass. The chromogenic substrate solution includes ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), TMB (tetramethyl benzidine), and the like, It is not limited thereto.

As used herein, the term "diagnosis marker", "diagnostic marker", "diagnosis marker" or "diagnosis marker" refers to a substance capable of diagnosing cancer cells by distinguishing them from normal cells. The marker may be an autoimmune antibody that specifically binds to BRD2 or a fragment comprising a complementarity determining region thereof.

As an analysis method for measuring the expression level of the antibody or protein fragment, Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue Immunostaining method, immunoprecipitation assay, complement fixation assay, flow cytometric analysis, protein chip assay, etc., but are not limited thereto, and specifically, the kit of the present invention comprises an anti-BRD2 autoimmune antibody or its complementarity determining region. It may be a kit using an ELISA coated with the BRD2 antigen protein, which is an epitope mimic that exhibits a specific reaction to a fragment containing

ELISA may be used to measure the protein expression level. ELISA is a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, an indirect ELISA using a labeled antibody recognizing a capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, Direct sandwich ELISA using another labeled antibody that recognizes antigen in a complex of antibody and antigen It may include various methods such as indirect sandwich ELISA using a secondary antibody.

For example, in the present invention, after attaching an antibody to a solid support and reacting the sample, a labeled antibody recognizing the antigen of the antigen-antibody complex is attached to enzymatically develop color or to the antibody recognizing the antigen of the antigen-antibody complex. Anti-BRD2 autoimmune antibody can be detected by a sandwich ELISA method in which color is developed enzymatically by attaching a secondary antibody labeled against it ( FIGS. 19 to 21 ). In addition, by checking the expression level of the fragment including the antigenic determinant of BRD2, it is possible to determine whether liver cancer occurs.

In a specific embodiment of the present invention, as a result of diagnosing liver cancer according to the above-described method, it was confirmed that a liver cancer individual and a normal individual could be distinguished and diagnosed with a sensitivity of 96.67% and a specificity of 90.00% (FIG. 22).

As another example of the analysis method, a Western blot using one or more antibodies to the diagnostic marker may be used. The total protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to a nitrocellulose membrane to react with the antibody. By confirming the amount of the generated antigen-antibody complex using a labeled antibody, the amount of the protein generated by gene expression can be checked to determine whether liver cancer occurs.

As another example, immunohistochemistry using one or more antibodies to the diagnostic marker may be performed. After collecting and fixing tissue from a subject having or suspected of having liver cancer, a paraffin format block is prepared by a method well known in the art. These are made into sections with a thickness of several μm and attached to a glass slide, and then the autoimmune antibody of the present invention or a fragment containing the complementarity determining region thereof is reacted with each other by a known method. Thereafter, the unreacted antibody or fragment containing the complementarity determining region thereof is washed, labeled with one of the above-mentioned detection labels, and whether the antibody or the fragment containing the complementarity determining region is labeled under a microscope is read. You can check if you have an infection.

As another example, a protein chip in which one or more antibodies to the diagnostic marker are arranged at a predetermined position on a substrate and immobilized at a high density may be used. In a method of analyzing a sample using a protein chip, a protein is separated from a sample and the isolated protein is hybridized with a protein chip to form an antigen-antibody complex and read it to check the presence or expression level of the protein, thereby causing the onset of liver cancer can check whether

According to the method of the present invention, when detecting an autoimmune antibody that specifically binds to BRD2 or a fragment comprising a complementarity determining region thereof or measuring its expression level, the BRD2 expression level is indirectly measured from a sample isolated from a suspected liver cancer individual. It is possible to predict not only whether the subject will develop liver cancer, but also whether the prognosis of the subject will be good in the future.

Another aspect of the present invention provides information for diagnosing liver cancer, comprising the step of detecting a fragment comprising an autoimmune antibody or its complementarity determining region that specifically binds to BRD2 using the composition of the present invention provide a way

Specifically, the method

(a) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 or a fragment comprising a complementarity determining region thereof from a biological sample isolated from a subject suspected of liver cancer; and

(b) the expression level of the autoimmune antibody or a fragment comprising the complementarity determining region measured in step (a), which is measured from a biological sample isolated from a normal subject, is an autoimmune antibody or its It may include comparing the expression level of the fragment containing the complementarity determining region.

The terms used herein are the same as described above.

The step (a) may be to measure the expression level of an autoimmune antibody that specifically binds to BRD2 or a fragment comprising a complementarity determining region thereof from a biological sample isolated from a subject suspected of liver cancer.

The sample may be a sample selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid, intercellular fluid, and combinations thereof isolated from an individual, but is not limited thereto.

As used herein, the term "subject" includes, without limitation, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish and birds. and may mean any animal (eg, human). More broadly, it may include, without limitation, the cell line of the animal.

The expression level of the autoimmune antibody or the fragment containing the complementarity determining region thereof was determined by Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue Immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometric analysis, protein chip assay, and any one or more methods selected from the group consisting of a combination thereof may be used for measurement, but limited to the above example It is not, and it is the same as described above.

In the step (b), the expression level of the autoimmune antibody or a fragment comprising the complementarity determining region measured in the step (a) is specifically binding to BRD2, which is measured from a biological sample isolated from a normal subject. It may be compared with the expression level of an antibody or a fragment comprising a complementarity determining region thereof.

In the present invention, the normal subject may be a sample derived from a subject in which the expression of anti-BRD2 autoimmune antibody is lower than that of a subject with or suspected of having liver cancer.

Another aspect of the present invention comprises the steps of (i) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 from an individual; (ii) administering a candidate substance for the treatment of liver cancer to the subject; (iii) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 from the subject to which the candidate substance has been administered; and (iv) when the expression level measured in step (iii) is reduced compared to the expression level measured in step (i), determining the candidate substance as a liver cancer treatment agent. provides

The terms used herein are the same as described above.

The step (i) may be to measure the expression level of an autoimmune antibody that specifically binds to BRD2 from an individual.

The measurement method is Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Octeroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry. Any one or more methods selected from the group consisting of (flow cytometric analysis), protein chip analysis, and combinations thereof may be used, but the present invention is not limited thereto, and this is the same as described above.

Step (ii) may be to administer a candidate substance for the treatment of liver cancer to the individual.

As used herein, the term "candidate for liver cancer treatment" refers to a substance expected to treat liver cancer, and any substance expected to improve or improve liver cancer directly or indirectly can be used without limitation. As an example, the candidate substance may include all therapeutic potential substances such as compounds, genes, or proteins.

Step (iii) may be to measure the expression level of an autoimmune antibody that specifically binds to BRD2 from an individual to which the candidate substance is administered.

The measurement method is the same as described above.

In step (iv), when the expression level measured in step (iii) is reduced compared to the expression level measured in step (i), the candidate substance may be determined as a liver cancer therapeutic agent.

Specifically, while checking the expression level of BRD2 before and after administration of the candidate substance, when the expression level is reduced compared to before administration of the candidate substance, the candidate substance may be determined as a therapeutic agent for liver cancer.

The composition for diagnosing liver cancer including a polypeptide including an antigenic determinant for detection of a Bromodomain-containing protein 2 (BRD2)-specific autoimmune antibody (auto-antibody) according to the present invention and a method for diagnosing liver cancer using the same do not require biopsy It is possible to diagnose liver cancer only by using non-invasive biological samples such as blood, plasma, serum, and lymph. In addition, the method is useful in diagnosing liver cancer, since the sensitivity and specificity for detecting the autoimmune antibody are remarkably high.

1 illustrates the process of constructing a cancer-associated autoimmune antibody (autoantibody)-producing B cell hybridoma library using HBx transgenic mice, which are liver cancer model mice. In HBx transgenic mice, liver cancer naturally develops at the age of 10 to 13 months. The spleen of a mouse with liver cancer was collected to collect B lymphocytes, and fused with mouse myeloma cells to construct a B cell hybridoma library. Antibodies secreted from each hybridoma were checked for reactivity against HepG2 cells, which are human liver cancer cells, and B cell hybridomas secreting liver cancer-related autoimmune antibodies were selected. Analysis was performed to present autoimmune antibody liver cancer biomarkers.
2 is a result of confirming the expression of the anti-XC246 autoimmune antibody antigen in various types of cancer cells including liver cancer by flow cytometric analysis. Cancer cells were immobilized and treated with anti-XC246 antibody after permeabilization of the cell membrane, and the secondary antibody conjugated with a fluorescent label was measured by flow cytometry after reaction. Anti-actin antibody response to the same cells was also measured, and anti-XC246 antibody expression was expressed based on the actin response.
3 is a result of intracellular antigen staining using an anti-XC246 antibody. As a result of confirming the intracellular expression location of the antigen protein recognized by the anti-XC246 antibody by fluorescence staining, it was clearly confirmed in the cytoplasm of cancer cell lines HepG2, Hepa-1c1c7, and Chang.
4 is a result confirming that the subtype of the anti-XC246 antibody is IgM.
5 shows the results of purification of anti-XC246 antibody from XC246 hybridoma culture medium and analysis by SDS-PAGE. After purification, a high-purity antibody was obtained, and it was confirmed that the 75 kDa heavy chain protein of the IgM antibody was expressed.
6 is a Western blot result confirming the anti-XC246 autoimmune antibody-specific antigen protein. It was confirmed that the anti-XC246 antibody recognized a protein having a molecular weight of 100 kDa.
7 is a result of confirming the isoelectric point of the anti-XC246 autoimmune antibody-specific antigen by two-dimensional electrophoresis of the HepG2 cell lysate. It was confirmed that the anti-XC246 antibody recognized a protein spot having a molecular weight of about 100 kDa and a characteristic of an isoelectric point of 9 level.
8 shows the results of quantifying the HepG2 cell lysate and the HepG2 cell serum-free culture medium, adhering to the membrane by 10, 5, and 1 ug, and dot blotting using the anti-XC246 antibody. By confirming that the antigen was detected not only in the cell lysate but also in the cell culture, it was verified that the XC246 antigen was extracellularly secreted.
9 is a result of immunoprecipitation of the cancer cell lysate with an anti-XC246 antibody and SDS-PAGE for purification in order to identify the XC246 antigen protein. SNU638 gastric cancer cells overexpressing XC246 antigen by aminating anti-XC246 antibody to resin with EDC (1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride)-NHS (N-hydroxy succinimide) linker The lysate was mixed and then immunoprecipitated to partially purify the anti-XC246 antibody-binding antigen. The immunoprecipitated protein was developed on SDS-PAGE for purification, and protein identification was performed from the protein band corresponding to the XC246 antigen.
10 is a mass spectrometry result of an anti-XC246 antibody-specific antigen protein. As a result of analysis by ESI-TRAP mass spectrometry, EEF2, BRD2 and MYOC1 were selected as XC246 antigen candidates.
11 is an anti-XC246 antibody-specific antigen verification result identified using siRNA. The siRNA of three XC246 antigen candidates (EEF2, BRD2, and MYOC1) was injected into HepG2 cells, and the anti-XC246 antibody response to the cell lysate was confirmed 72 hours later. As a result, it was confirmed that BRD2 was the XC246 antigen, and the anti-BRD2 antibody response protein band was confirmed to match the XC246 antigen, thereby reconfirming that the XC246 antigen was BRD2.
12 is a Western blot showing the results of immunoprecipitation of HepG2 cell lysate using anti-XC246 antibody conjugation resin and confirming the anti-BRD2 antibody response. By confirming that the immunoprecipitated protein was detected with the anti-BRD2 antibody, it was reconfirmed that the antigen responding to the anti-XC246 antibody was BRD2.
13 is a result of expressing the BRD2 recombinant protein fused with Flag-tag in HEK293T cells, immunoprecipitation with anti-Flag antibody, and Western blotting with anti-XC246 antibody. Both Flag-BRD2 recombinant protein subtypes 1 and 3 were detected by the anti-XC246 antibody, reconfirming that BRD2 is an antigen of the anti-XC246 antibody.
14 is a result of screening an anti-XC246 antibody-specific epitope from a cyclic peptide library. Specific reaction phages were screened with anti-XC246 antibody from a phage library expressing random sequence cyclic peptides, and the result of sequential amplification of antibody-specific phages was shown while repeating panning.
15 shows the amino acid sequence of the anti-XC246 antibody-specific antigenic determinant expressed by the selected phages.
16 shows the results of confirming the anti-XC246 antibody reactivity of 10 selected phages by ELISA. It was confirmed that XC246p2, XC246p5, XC246p6 and XC246p9 phages had high anti-XC246 antibody-specific reactivity.
17 shows the selected epitope by modifying the cyclic structure of the XC246p2, XC246p5, XC246p6 and XC246p9 phage epitope showing high reactivity to the anti-XC246 antibody to a linear form and confirming the change in the reactivity of the anti-XC246 antibody. This is the result confirming that the anti-XC246 antibody response is structure-dependent.
18 is a result confirming that the selected phage competitively inhibits the cancer cell response of the anti-XC246 antibody, and confirms that the phage antigen sufficiently mimics the structure of the antigenic epitope in the cell.
19 shows the result of conjugating the sequence of the XC246p9 epitope having a cyclic structure to the BSA protein using a continuous linker of two miniPEG2s (8-Amino-3,6-Dioxa-Octanoic acid). For the conjugation reaction, the amine residue at the end of miniPEG and the carboxyl group of bovibe serum albumin (BSA) were used. Coomassie staining: CBB).
20 is a result of measuring the anti-XC246 antibody response to BSA-miniPEG-XC246p9. BSA-miniPEG-XC246p9 antigen was coated at various concentrations, and serially diluted anti-XC246 antibody was used as the primary antibody for ELISA analysis.
21 is a result confirming by competitive Western blot that the BSA-miniPEG-XC246p9 antigen mimics the intracellular antigen structure. Anti-XC246 antibody or anti-XC246 antibody pre-treated with BSA-miniPEG-XC246p9 was used for Western blot against HepG2 and Huh7 cell lysate, and it was confirmed that the antibody response was inhibited by treatment with BSA-miniPEG-XC246p9 antigen. It was verified that it mimics the antigenic structure.
22 is a result confirming that blood autoimmune antibody detection using BSA-miniPEG-XC246p9 antigen can be utilized for liver cancer diagnosis. Serum analysis of liver cancer patients (n=30) and normal people (n=30) was performed by ELISA using BSA-miniPEG-XC246p9 as a coating antigen. The serum response value was expressed as a comparison value (%) of the BSA-miniPEG-XC246p9 response value to the BSA-miniPEG response value. As a result, it was confirmed that when the cutoff value was set to 113, the reactivity to the liver cancer sample could be remarkably distinguished with a sensitivity of 96.67% and a specificity of 90.00%.
23 is a diagram showing the amino acid sequence of the heavy chain protein of the XC246 antibody.
24 is a diagram showing the amino acid sequence of the light chain protein of the XC246 antibody.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Confirmation of human cancer cell reactivity of HBx mouse-derived anti-XC246 autoimmune antibody (autoantibody)

In order to secure autoimmune antibodies generated along with cancer development, HBx transgenic mice, which have been verified to develop a form of liver cancer similar to that of human liver cancer, were used. Among HBx transgenic mice, spleen cells from 10 to 13-month-old individuals confirmed for liver cancer were obtained as a B cell group and cell fusion with Sp2/0, a mouse myeloma cell, to prepare a B cell hybridoma cell group (Fig. One). Cell fusion was performed according to a general B cell hybridoma preparation method and a HAT (hypoxanthine-aminopterin-thymidine medium) medium selection method. Only cloned cells were selectively cultured, and human cancer cell reactivity was confirmed with their cell culture medium, and only B cell hybridomas whose reactivity was confirmed were selected and maintained again.

In order to confirm the reactivity of the cancer model mouse-derived autoimmune antibody to human cancer cells, intracellular staining and flow cytometric analysis were performed on the fixed and permeabilized cancer cell lines.

Specifically, 70-80% confluence of mouse and human cancer cells (Hepa1c1c7, HeLa, SK-Hep1, Chang, A549, Hep3B, HepG2, LNCap/LN3, HT29, Huh7, MCF7, PLC/PRF5) cells After culturing in a culture plate in a state in which the grown cells are filled in the space for a certain period of time during culture), they were removed from the plate by treatment with trypsin and washed with phosphate buffered saline (PBS). The prepared cells were dispensed into tubes by 2X10 ⁵ each, and 100 μl of Cytofix/Cytoperm solution (BD Biosciences) was treated at 4° C. for 20 minutes to immobilize and permeabilize. After the reaction, 1 ml of Cytowash/Cytoperm solution (BD Biosciences) was added and mixed well, and then precipitated by centrifugation at 1,700 rpm for 5 minutes to wash the fixed cells. 50 μl of the primary antibody solution (XC246 B cell hybridoma cell culture medium or purified anti-XC246 antibody solution) was added to the washed cell sample and reacted at 4° C. for 40 minutes. After the reaction, cells were washed three times, and treated with anti-mouse antibody-FITC (anti-mouse Igs-FITC, DaKo Co., Ltd.) at 4° C. for 40 minutes. After the reaction, the cells were washed 3 times again, and the cell precipitate was dissolved in 300 μl of PBS and analyzed with FACScalibur (BD Biosciences). Mean fluorescence values corresponding to the antibody response were measured, and XC246 antigen expression was expressed as a relative value for the anti-actin antibody response.

As a result, it was confirmed that the XC246 antigen was expressed in Hepa1c1c7, a mouse liver cancer cell, and various human cancer cells, and the expression level was different from each other ( FIG. 2 ).

Next, in order to confirm the expression location of the XC246 antigen in cancer cells, intracellular staining using an anti-XC246 antibody was observed with a confocal fluorescence microscope.

Specifically, 1x10 ⁵ cells were cultured on a coverslip using a 6-well plate, washed once with PBS, and then treated with Cytofix/Cytoperm solution at room temperature for 20 minutes to immobilize and permeabilize. After the reaction, it was washed twice with a PBS washing solution containing 0.1% (w/v) BSA (bovine serum albumin) and 0.1% (w/v) saponin, and 1% (w/v) BSA was added. After treating the PBS blocking solution at room temperature for 30 minutes, anti-XC246 antibody at a concentration of 2 ug/ml was treated at room temperature for 1 hour. After the reaction, washing was performed again three times and reacted with an anti-mouse Igs-Rhodamine (anti-mouse Igs-Rhodamine, Abcam) solution at room temperature for 1 hour. Wash the cells 3 times again, and use 50 ul of a mounting solution (Vectashield) containing DAPI (4′,6-diamidino-2-phenylindole) to stain the nuclei. After immobilization, the intracellular location of the antigen was confirmed with a fluorescence microscope.

As a result, the antigen staining of the anti-XC246 antibody was strongly confirmed at the cytoplasmic location, confirming that the anti-XC246 antibody-reactive antigen was expressed in the cancer cell cytoplasm (FIG. 3).

From the above results, it was confirmed that the XC246 antigen was expressed in the cytoplasm of various cancer cell lines.

Example 2. Subtyping and purification of XC 246 antibody

The subtype of the anti-XC246 antibody was determined using a mouse antibody subtype kit (Isotyping Kit, Pierce Corporation). On an ELISA plate coated with a mouse antibody subtype (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM)-specific anti-mouse capture antibody, diluted 1/50 of the XC246 hybridoma culture in TBS (Tris-buffered saline) to 50 ul , and simultaneously treated with anti-mouse-IgG+IgA+IgM-HRP (horseradish peroxidase) antibody to induce a reaction for 1 hour and then use TMB (3, 3', 5, 5'-Tetramethyl-benzidine) A color reaction was performed.

As a result, it was confirmed that the anti-XC246 antibody subtype is the IgM type ( FIG. 4 ).

Next, the anti-XC246 antibody identified as IgM type was purified from the hybridoma culture using agarose beads conjugated with Protein-L. The purified antibody was used for subsequent analysis after quantification with Bradford reagent. The purified antibody sample was developed on 10% SDS-PAGE after treatment with a reducing reagent, and Coomassie staining (CBB) was performed.

As a result, it was confirmed that the IgM type antibody was purified with high purity, and the heavy chain protein (SEQ ID NO: 11) and light chain protein (SEQ ID NO: 12) of the IgM type were confirmed (FIG. 5).

The B cell hybridoma cell line producing the anti-BRD2 autoimmune antibody was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on November 23, 2020, and was given an accession number KCTC 14386BP. Specifically, the hybridoma cell line may be a cell line with accession number KCTC 14386BP.

Example 3. Confirmation of expression of anti-XC246 antibody-specific antigen in various cancer cells

The expression of anti-XC246 antibody-specific antigen protein was confirmed in human cancer cell lines through Western blot.

Specifically, the target cancer cells (HepG2, Hep3B, Huh7, SNU638, A549, HT29, LNCap/LN3) are cultured and then collected and collected with NP40 buffer [NP40 buffer: PBS containing 1.0 % (v/v) NP40, protease inhibitor cocktail ( Roche)] and used as a protein analysis sample.

Protein quantification of the cell lysate was performed by the Bradford method. The prepared protein samples were developed in 50 ug each under reducing 10% SDS-PAGE conditions, and transferred to a PVDF (Polyvinylidene fluoride) membrane. The membrane to which the protein has been transferred is blocked by immersing it in a TBS solution containing 5% (w/v) skim milk, and the purified anti-XC246 antibody is diluted in a blocking solution to a concentration of 0.5 ug/ml. Primary antibody was treated. After primary antibody treatment, wash thoroughly with TBST (TBS containing 0.1% (v/v) tween-20), and after treatment with anti-mouse IgGAM-HRP with secondary antibody, Enhanced Chemi-Luminescence (ECL) Antibody-reactive protein bands were identified by the method.

As a result, in human cancer cell lines, the anti-XC246 antibody-specific antigen protein was identified as a band having a molecular weight of about 110 kDa ( FIG. 6 ). In addition, it was confirmed that there is a difference in the expression level depending on the cancer cell line.

Next, two-dimensional gel electrophoresis (2D-PAGE) was performed for isoelectric point analysis of the antigen recognized by the anti-XC246 antibody. After developing a HepG2 cell urea lysate on an IPG (immobilized pH gradient) strip with a pH of 3 to 10, the protein is divided according to the isoelectric point, and the IPG strip is again developed on 6% SDS-PAGE. Western blot was performed to confirm the antigen's isoelectric point.

As a result, an anti-XC246 antibody staining spot having an isoelectric point of about 9 was confirmed in the vicinity of ~110 kDa (FIG. 7).

In order for an intracellular antigen to induce a specific antibody, extracellular exposure of the antigen is essential. Therefore, in order to check whether the XC246 antigen is extracellularly secreted, HepG2 cells in a 50% confluence state were cultured for 3 days, and then the cell culture medium (CCM) was concentrated, and on a nitrocellulose membrane. After adhesion, dot blotting was performed by blocking the membrane in the same manner as Western blot to perform anti-XC246 antibody reaction. NP40 lysate of HepG2 cells was used as an antigen-positive sample, and BSA was used as a non-reactive sample.

As a result, it was confirmed that the antigen of the anti-XC246 antibody was detected not only in the cell lysate but also in the cell culture medium, thereby verifying the extracellular secretion characteristic of the anti-XC246 antibody antigen ( FIG. 8 ).

Example 4. Identification of anti-XC246 antibody specific antigen protein

To identify the anti-XC246 antibody-specific antigen protein, the antigen protein was immunoprecipitated with the anti-XC246 antibody and then analyzed by mass spectrometry.

Specifically, a resin conjugated with anti-XC246 antibody was prepared for antigen immunoprecipitation. A PBS solution containing 1 mg of anti-XC246 antibody was mixed with resin (Thermo) with EDC (1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride)-NHS (N-hydroxy succinimide) linker and 2 at room temperature. By reacting with time, amine conjugation of the antibody and the resin was induced, and after washing with PBS 5 times, the reaction was terminated by treatment with 1M Tris-HCl, pH 7.4 solution to terminate the reaction with the anti-XC246 antibody-conjugated resin (XC246). -resin) was prepared.

Antigen-antibody binding was induced by mixing 9.69 mg of gastric adenocarcinoma (SNU638) NP40 lysate with the prepared anti-XC246 antibody conjugation resin and mixing well at 4°C for 16 hours.

After the antibody-antigen reaction, the resin was sufficiently washed with NP40 buffer, 600 ul of SDS-PAGE sample buffer was added, and an immunoprecipitated protein sample containing antigen was prepared by heating at 100°C. Protein samples were divided in a volume ratio of 10:90 and developed in different wells under reducing 12% SDS-PAGE conditions. SDS-PAGE gel pieces with 10% protein were transferred to a PVDF membrane and Western blot with anti-XC246 antibody. to confirm the location of the antigen protein band. SDS-PAGE gel pieces in which 90% protein was developed were stained with Coomassie seeds, and a protein band fragment at the same position as the protein position confirmed by Western blot was used for mass spectrometry.

In order to exclude non-specific proteins from mass spectrometry, the same amount of control resin to which the anti-XC246 antibody was not conjugated was used in the entire process above. An in-gel digestion reaction using a trypsin protease was performed on the prepared protein band fragment (FIG. 9). Information on the protein sequence was obtained by mass spectrometry using ESI-TRAP mass spectrometry for peptide fractions extracted from each protein band after in-gel digestion.

As a result, BRD2 (Bromodomain-containing protein 2), MYO1C (Isoform 3 of Unconventional myosin-Ic), and protein bands 1 and 2, which have a protein molecular weight of around 10 kDa and a protein isoelectric point value of around 9, have the highest score in common. The identified EEF2 (Elongation factor 2) was selected as a candidate protein for the anti-XC246 antibody-specific antigen ( FIG. 10 ).

Next, in order to verify the identification results of the proteins (EEF2, MYC1C and BRD2) selected as candidate proteins for the anti-XC246 antibody-specific antigen, siRNA (Bioneer) that inhibits the expression of EEF2, MYC1C and BRD2 was After injection into HepG2 cells using RNAiMax (Invitrogen), it was checked whether the anti-XC246 antibody response was reduced.

Cells were harvested 72 hours after siRNA treatment and the expression of the corresponding gene was confirmed by RT-PCR. As a result, it was confirmed that the expression of each gene was suppressed by 50% or more.

At the same time, NP40 buffer lysate of siRNA-injected cells was developed by reducing 12% SDS-PAGE and then Western blot treated with anti-XC246 antibody was performed. Thus (Fig. 11), it was estimated that the anti-XC246 antibody-specific antigen protein was BRD2.

To verify this again, the same cell lysates were analyzed with an anti-BRD2 antibody, and it was confirmed that the BRD2 antigen reacted in the same manner as the XC246 antigen reaction, thereby confirming that the anti-XC246 antibody-specific antigen protein was BRD2 ( FIG. 11 ).

Next, it was reconfirmed that BRD2 was an anti-XC246 antibody-specific antigen protein through immunoprecipitation using XC246-resin and expression of BRD2-Flag protein.

Specifically, XC246-resin was prepared by the method of Example 4, and antigen-antibody binding was induced while mixing 600 ug of HepG2 lysate with the resin at 4° C. for 16 hours. After the reaction, the resin was sufficiently washed with NP40 solution and eluted by treatment with SDS-PAGE sample buffer. The obtained immunoprecipitated protein sample was developed on 12% SDS-PAGE, and Western blot was performed using an anti-BRD2 antibody.

As a result, it was confirmed that the protein immunoprecipitated with the anti-XC246 antibody was BRD2, and it was reconfirmed that the anti-XC246 antibody-specific antigen protein was BRD2 (FIG. 12).

Next, a vector expressing the Flag-tag fusion BRD2 recombinant protein was constructed using cDNA of the HepG2 hepatocellular carcinoma cell line. BRD2 subtypes 1 and 3 were constructed, and Flag-BRD2 subtypes 1 and 3 expression vectors were injected into HepG2 cells using PEI (Polyethylenimine). After 72 hours, cells were harvested, disrupted in NP40 buffer, immunoprecipitated using anti-Flag antibody, and Western blot was performed using anti-XC246 antibody.

As a result, it was confirmed that the anti-XC246 antibody binds to both subtypes of BRD2 ( FIG. 13 ).

Through the above results, it was re-verified that the specific antigen of the anti-XC246 autoimmune antibody was BRD2.

Example 5. Anti-XC246 antibody specific reaction epitope screening

Based on the similarity of the cancer-generating characteristics of the HBx liver cancer model mouse to that of human liver cancer, when an antibody having similar characteristics to the cancer model mouse-derived autoimmune antibody XC246 identified in Example 4 is induced in a liver cancer patient, it can be used as a cancer biomarker. assumed to be possible. However, BRD2, which has been identified as an anti-XC246 autoimmune antibody-specific antigen, has a molecular weight of 100 kDa, and it is expected that it will be difficult to secure the recombinant protein in a form expressed in the human body. It was intended to be derived and used as an antigen for antibody detection.

Specifically, the antigen structure specific to the anti-XC246 antibody was screened from a random cyclic peptide expression library (Ph.D.-C7C Phage Display Peptide Library kit; New England Biolabs). As a peptide expression library, a cyclic peptide expression phage library (Ph.D.-C7C Phage Display Peptide Library) in which 7 amino acids are expressed in a random sequence and a total of 9 amino acids including cysteine residues at both ends form a cyclic structure kit; New England Biolabs) was used, and the panning process was performed according to the method suggested by the manufacturer. 1 ug of anti-XC246 antibody was mixed with phage virions expressing 2x10 ¹¹ different peptides and 200 ul TBST solution, reacted at room temperature for 20 minutes, and pre-blocking solution [0.1 M NaHCO ₃ , pH 8.6, 5 mg/ml BSA, 0.02% (w/v) NaN ₃ ] was mixed with 25 ul of treated protein L-agarose beads and reacted at room temperature for 15 minutes. Antibody-reacted phages are precipitated by centrifugation and recovered in the form of an antibody-virion-protein L-agarose conjugate, washed several times with TBST, and then a pH 2.3 eluate (0.2 M Glycine-HCl, pH 2.3, 1 mg/ml BSA) was treated with 1 ml and eluted. Then, a 1 M Tris-HCl solution at pH 9.1 was quickly added to bring it to neutral pH.

Some of the eluted phages were used to titration the number of phages, and the rest was used to amplify the phages. For the amplified phage, panning was performed again in the above-described manner.

As a result, it was confirmed that the number of phages reacting with the antibody increased during the 4 panning process for the anti-XC246 antibody, confirming that the antibody-specific reactive phages were amplified (FIG. 14), and the phages obtained after panning 4 times The epitope sequences of 10 randomly selected phages were determined by DNA sequencing to obtain 10 different polypeptide sequences (FIG. 15).

Next, in order to confirm the reactivity of the phages to the anti-XC246 antibody, ELISA was performed using the phage as a coating antigen and the anti-XC246 antibody as the primary antibody.

Specifically, the selected phages were amplified and partially purified by treatment with a PEG/NaCl solution to be used as an ELISA-coated antigen. 10 ¹⁰ pfu of purified phage was diluted in 100 ul of coating solution (0.1 M sodium bicarbonate buffer, pH 8.6) and added to each well of a 96-well Maxisorp ELISA plate. Plates with phage added for antigen coating were stored at 4° C. for at least 16 hours. After phage coating, 300 ul of skim milk solution [5% (w/v) skim milk/TBST] was added and reacted at room temperature for 1 hour to block the remaining area after antigen coating. After blocking, 100 ng/100 ul of anti-XC246 antibody and non-specific XC90 and K94 antibodies were added and reacted at room temperature for 90 minutes. After the reaction, it was washed 6 times with TBST, and a secondary antibody, anti-mouse IgGAM-HRP (Pierce), was diluted at a ratio of 1:2500 and treated. The secondary antibody was also reacted at room temperature for 90 minutes and then washed 6 times with TBST. After color reaction was carried out using TMB solution (Pierce) as the HRP substrate of the secondary antibody, absorbance was measured at 450 nm to quantify the antigen-antibody reaction.

As a result, all phages used as antigens showed reactivity to the anti-XC246 antibody (FIG. 16).

Next, XC246p2, XC246p5, XC246p6, and XC246p9 phages, which previously showed high reactivity to the anti-XC246 antibody, were treated with DTT (Dithiothreitol), a reducing agent, at 56 ° C. for 30 minutes. A linear change of the cyclic peptide structure was induced through alkylation of the cysteine residue by breaking the disulfide bond and treating with Iodoacetamide (IAA).

Each well of a 96-well Maxisorp ELISA plate by diluting the prepared linear structural epitope phage and the phage expressing the conventional cyclic structure in 100 ul of a coating solution (0.1 M sodium bicarbonate buffer, pH 8.6) of 10 ¹⁰ pfu each. After coating, the anti-XC246 antibody was used as a primary antibody and the reactivity was compared by ELISA.

As a result, all of the antigenic determinants presented by the four phages (XC246p2, XC246p5, XC246p6, XC246p9) selected to show high reactivity to the anti-XC246 antibody exhibited anti-XC246 antibody binding ability that was more circular structure-dependent than sequence specificity. It was confirmed that it was visible (FIG. 17).

Example 6. Competitive Inhibition of Anti-XC246 Antibody to Hepatocarcinoma Response Using Selected Phage

Anti-XC246 antibody-specific phage when the reaction of the anti-XC246 antibody with the cell-expressed antigen protein is measured by flow cytometry to confirm that the antibody-specifically selected phages sufficiently mimic the antigenic determinant of the cancer cell-expressing antigen protein. was added to confirm that the reaction was competitively inhibited. Flow cytometry was performed in the same manner as in Example 1, in the case of treating only the anti-XC246 antibody as the primary antibody, and the anti-XC246 antibody with four selected phages (XC246p2, XC246p5, XC246p6, XC246p9) A case of pre-mixing and reacting for 1 hour was compared. 100 ng of the antibody was used and 10 ¹¹ or 10 ¹² pfu of the phage were used. As a negative control, XC90p2 phage expressing a cyclic peptide of a different sequence was used, and the antibody-antigen reaction was confirmed in HepG2.

As a result, as shown in FIG. 18 , it was confirmed that the selected phages inhibited the response of the anti-XC246 antibody to HepG2 cells by 90% or more.

From the above results, it can be seen that the epitope of the cyclic peptide epitope of the selected phages sufficiently mimics the epitope of the anti-XC246 antibody-specific antigen protein.

Example 7. Preparation of XC246p9 epitope presentation vehicle for autoimmune antibody detection

In order to secure an antigen for autoimmune antibody detection, a cyclic XC246p9 sequence that sufficiently mimics the specific reaction structure of an anti-XC246 antibody was conjugated to BSA using miniPEG2 as a linker to prepare an antigen.

Specifically, the XC246p9 peptide (miniPEG22-XC246p9) including miniPEG2, a linker for minimizing the interaction between the protein carrier and the peptide, was synthesized by Peptron Co., Ltd. BSA (Thermo), a carrier protein, was dissolved in activation buffer [0.1M MES (2-[morpholino]ethanesulfonic acid), 0.5M NaCl, pH 6.0] at a concentration of 10 mg/mL, EDC and final Inducing the carboxy terminus of BSA to have a semi-stable amine-reactive sulfo-NHS ester structure by mixing with a concentration of 5 mM sulfo-NHS (sulfo-NHS) and reacting at room temperature for 15 minutes did. After completion of the reaction, 1M sodium phosphate dibasic was added to the reaction solution to adjust the pH to 7.0 or higher for the peptide conjugation reaction, and 1 mg of miniPEG2-XC246p9 peptide dissolved in high-purity water was added to the reaction solution for 2 hours at room temperature. Mixing induced BSA-miniPEG2-XC246p9 peptide amine conjugation. Thereafter, the reaction was terminated by adding a Tris-HCl (pH 7.4) solution to a final concentration of 50 mM.

The BSA-miniPEG2-XC246p9 conjugated protein solution after the reaction is injected into a Zeba™ Spin Desalting column (7K MWCO, Thermo) and centrifuged at 1,000 g for 2 minutes to remove the unconjugated residual peptide. The XC246p9 antigen was prepared (FIG. 19).

To test the antibody reaction activity of the prepared BSA-miniPEG2-XC246p9 antigen, the BSA-miniPEG2-XC246p9 antigen was serially diluted 1/5 at 0.5 ug/well and added to each well of a 96-well Maxisorp ELISA plate. After antigen coating at 4° C. for 16 hours, washing twice with TBST, and blocking by adding 300 ul of 5% skim milk. Anti-XC246 antibody was prepared by sequentially diluting 1/3 at 400 ng/well in a blocking solution, and added to the blocking plate to induce an antigen-antibody reaction at 37° C. for 2 hours. After completion of the reaction, 100 ul of anti-mouse-IgG-HRP (CST) diluted by 1/2500 in blocking buffer was added after washing 4 times with TBST to remove excess antibody, followed by secondary antibody reaction for 90 minutes. After the reaction, the reaction was again washed 4 times with TBST to remove the non-reacted residual secondary antibody, 100 ul of TMB was added to generate reaction for 10 minutes, and the reaction was terminated by adding the same amount of 0.1M sulfuric acid (H ₂ SO ₄ ) solution. The antigen-antibody reaction was quantified by measuring the absorbance at OD450nm (FIG. 20).

As a result, BSA-miniPEG2-XC246p9, a synthetic peptide epitope presenting vehicle suitable for detecting cancer-related autoimmune antibody XC246, was obtained.

Next, to determine whether the BSA-miniPEG2-XC246p9 antigen sufficiently mimics the antigenic determinant of the cancer cell-expressed antigen protein, the reaction of the anti-XC246 antibody with the cell-expressed antigen protein was measured by Western blot. BSA-miniPEG2-XC246p9 It was confirmed whether the response of the anti-XC246 antibody to the intracellular antigen was competitively inhibited by the addition of the antigen. Western blotting was performed in the same manner as in Example 3, in the case where only the anti-XC246 antibody was treated as the primary antibody, and the anti-XC246 antibody was premixed with the BSA-miniPEG2-XC246p9 antigen and reacted for 1 hour. treated cases were compared. The antibody was used at a concentration of 0.1 ug/ml, and the BSA-miniPEG2-XC246p9 protein was used at a concentration of 0.3 ug/ml. As a negative control, BSA-miniPEG2-XC90p2 expressing cyclic amino acids of different sequences and BSA-miniPEG2 protein having only a miniPEG2 linker were used, and responses were confirmed in HepG2 and Huh7 cells.

As a result, as shown in FIG. 21 , it was confirmed that the BSA-miniPEG2-XC246p9 antigen mostly inhibited the response of the anti-XC246 antibody to the antigens expressed in HepG2 and Huh7 cells.

From the above results, it can be seen that the BSA-miniPEG2-XC246p9 antigen sufficiently mimics the antigenic determinant of the anti-XC246 antibody-specific antigen protein.

Example 8. Diagnosis of liver cancer using XC246p9 epitope expressing antigen

Based on the results of Example 7, an ELISA was prepared using the BSA-miniPEG2-XC246p9 antigen (recombinant protein) as a blood anti-BRD2 autoimmune antibody detection antigen.

Specifically, 0.1 ug of BSA-miniPEG2-XC246 antigen was diluted in 100 ul of PBS, pH 7.4 solution and then added to each well of a 96-well Maxisorp ELISA plate, followed by coating at 4° C. for 16 hours. At this time, in order to exclude a non-specific reaction of human serum to BSA, an equal amount of BSA-miniPEG2, which does not have an anti-XC246 antibody-specific XC246p9 cyclic sequence, was coated. After coating, the excess antibody was removed and a blocking buffer in which 0.1% polyvinyl alcohol (Molecular weight 31-50K, Sigma) was dissolved in TBST buffer was added to each well by 300 μl, followed by blocking at room temperature for 2 hours. After blocking, serum derived from liver cancer patients and normal persons inactivated at 56° C. for 30 minutes was diluted 1/50 in a blocking buffer containing 5% BSA and treated with a primary antibody. Serum derived from normal humans and serum from liver cancer patients were purchased from the Korea Human Resources Bank and used (Provided No.: 09SA2020010-001). The primary antibody reaction was carried out with stirring at 37° C. for 2 hours, and after the reaction, excess antibodies were washed 4 times with TBST. As a secondary antibody, 100 ul of anti-human IgGAM-HRP (anti-human IgGAM-HRP, Pierce Corporation) was diluted in a blocking solution at a ratio of 1/2000 and reacted at 37° C. for 90 minutes. After the reaction, it was washed again with TBST 4 times, and 100 ul of TMB solution was added to proceed with HRP reaction, and then absorbance was measured at 450 nm to quantify the amount of anti-XC246p9 (anti-BRD2) antibody in human serum.

As a result, sera of normal subjects and liver cancer patients were distinguished with a sensitivity of 96.67% and a specificity of 90.00% (p<0.0001; AUC=0.9388) ( FIG. 22 ).

From the results of the above Examples, the anti-BRD2 autoimmune antibody according to the present invention can be used as a liver cancer biomarker, and the antigenic determinant mimetic exhibiting a specific response to the anti-BRD2 autoimmune antibody is applied to ELISA to diagnose liver cancer It was confirmed that it can be provided as

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Korea Institute of Bioscience and Biotechnology KCTC14386BP 20201123

<110> Korea Research Institute of Bioscience and Biotechnology <120> Anti-bromodomain-containing protein 2-specific autoantibody cancer biomarker, antigen composition for detection thereof, and liver cancer diagnosis method using the same <130> KPA210093-KR <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 1 <400> 1 Ser Ser Met Phe Leu Pro Ser 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 2 <400> 2 Ser Ser Gln Trp Leu Pro Phe 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 3 <400> 3 Thr Ser Ala Leu Phe Pro Trp 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 4 <400> 4 Val Ser Ala Ser Phe Pro Phe 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 5 <400> 5 Asn Gln Val Ala Tyr Pro Trp 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 6 <400> 6 Phe Ser Ala Leu Tyr Pro Trp 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 7 <400> 7 Cys Arg Leu Arg Trp Pro His 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 8 <400> 8 Thr Ser Ser Phe Phe Pro His 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 9 <400> 9 Thr Ser Val Phe Leu Pro His 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> clone 10 <400> 10 Ser Thr Ala Met Ala Leu Val 1 5 <210> 11 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain protein <400> 11 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser His Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Ser Asp Trp Glu His Trp Gly Gln Asp Thr Thr Leu Thr Val Ser 100 105 110 Ser Glu Ser Gln Ser Phe Pro Asn Val 115 120 <210> 12 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> light chain protein <400> 12 Asp Ile Val Leu Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Ser His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser 115 120

Claims (17)

A fragment comprising an autoimmune antibody or complementary determining regions (CDR) thereof that specifically binds to Bromodomain-containing protein 2 (BRD2).
The method of claim 1, wherein the autoimmune antibody recognizes a polypeptide consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10, comprising an autoimmune antibody or a complementarity determining region thereof snippet.
A hybridoma cell line producing the autoimmune antibody of any one of claims 1 to 2.
The cell line according to claim 3, wherein the hybridoma cell line is Accession No. KCTC 14386BP.
Claims 1 to 2, which specifically binds to the autoimmune antibody of any one of claims 1 to 2, and consists of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10, epitope poly peptide.
The polypeptide according to claim 5, wherein the epitope polypeptide further comprises cysteines at both ends.
A composition for diagnosing liver cancer, comprising an agent for measuring the expression level of the autoimmune antibody of any one of claims 1 to 2 or a fragment comprising a complementarity determining region thereof.
The composition of claim 7, wherein the agent for measuring the expression level of the autoimmune antibody or a fragment comprising a complementarity determining region thereof is a polypeptide that specifically binds to the autoimmune antibody.
The composition of claim 8, wherein the polypeptide comprises an antigenic determinant consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10.
The composition of claim 9, wherein the polypeptide further comprises cysteines at both ends.
A kit for diagnosing liver cancer comprising the composition of claim 7.
12. The method of claim 11, wherein the kit comprises Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement The kit, which uses any one or more methods selected from the group consisting of a fixed assay, a flow cytometric analysis, a protein chip assay, and a combination thereof.
A method of providing information for diagnosing liver cancer, comprising detecting a fragment comprising an autoimmune antibody or its complementarity determining region that specifically binds to BRD2 using the composition of claim 7 .
14. The method of claim 13, wherein the method
(a) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 or a fragment comprising a complementarity determining region thereof from a biological sample isolated from a subject suspected of liver cancer; and
(b) the expression level of the autoimmune antibody or a fragment comprising the complementarity determining region measured in step (a), which is measured from a biological sample isolated from a normal subject, is an autoimmune antibody or its Comprising the step of comparing the expression level of the fragment comprising the complementarity determining region, the method.
The method of claim 13, wherein the sample is a sample selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid, intercellular fluid, and combinations thereof isolated from a subject.
14. The method of claim 13, wherein the expression level of the autoimmune antibody or a fragment comprising a complementarity determining region thereof is determined by Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmune diffusion method, Oukteroni immune diffusion method, It is measured using any one or more methods selected from the group consisting of rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometric analysis, protein chip assay, and combinations thereof, Way.
(i) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 from a non-human subject;
(ii) administering a candidate substance for the treatment of liver cancer to the subject;
(iii) measuring the expression level of an autoimmune antibody that specifically binds to BRD2 from the subject to which the candidate substance has been administered; and
(iv) when the expression level measured in step (iii) is reduced compared to the expression level measured in step (i), determining the candidate substance as a liver cancer treatment agent.

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