KR101439856B1 - A marker comprising anti-ATIC autoantibodies and a composition comprising antigen thereof for diagnosing liver cancer - Google Patents

Abstract

The present invention relates to a composition for diagnosing liver cancer comprising an autoantibody specifically binding to ATIC or a fragment containing an antigen-binding site (paratope) thereof and an agent for measuring the expression level of the fragment, A hybridoma cell line producing the autoimmune antibody, a liver cancer diagnostic kit containing the composition, a method of providing information for diagnosis of liver cancer using the composition, and a screening method of a therapeutic agent for liver cancer using the autoimmune antibody. When the anti-AIC specific autoimmune antibody of the present invention is used as a liver cancer diagnostic marker, non-invasive biological samples such as blood, plasma, serum, and lymphatic fluid are used to detect about 87% sensitivity and about 88% And can easily diagnose liver cancer using only the amino acid sequence identified in the present invention, which is effective in developing a kit for the diagnosis of liver cancer.

Description

[0001] The present invention relates to a diagnostic marker for hepatocellular carcinoma comprising an anti-AIC autoimmune antibody and a composition for diagnosing liver cancer comprising the same,

The present invention relates to an autoantibody specifically binding to ATIC or a fragment containing an antigen-binding site (paratope) thereof, an antigen fragment specifically binding to the autoimmune antibody, A hybridoma cell line producing the autoimmune antibody, and a kit for the diagnosis of liver cancer comprising the composition of the present invention. The present invention also relates to a method for providing information for diagnosis of liver cancer using the composition of the present invention and a screening method for treating liver cancer using the autoimmune antibody.

Liver disease, including hepatitis, liver cirrhosis, and liver cancer, is the most common single disease in Korea, Japan, Taiwan, China, and most Southeast Asian countries. Liver cancer, 9.2%) (Ministry of Health, Welfare and Family Affairs, 2007), and the death rate of liver cancer among Korean men is the top in the world.

To date, liver biopsy has been conducted to detect liver cancer. In addition, several biomarkers have been known for such diagnosis, prognostic evaluation, or therapeutic evaluation. Of these, AFP and PIVKA-II are the most well known markers, but they still have limitations in terms of specificity and sensitivity. Recently, several kinds of candidate cancer marker proteins and genes have been reported by genomics and proteomics studies. However, the reported genes are genes mainly for tissues, and the possibility of blood secretion and diagnosis of blood It is still not clear. This is due to the intrinsic nature of the biomarkers, most tumor markers are being explored centering on differences in gene expression in tissues, and because of the nature of biomarkers, high expression of genes in tissues is not diagnostic of urine or serum . Therefore, for the convenience of diagnosis, it is important to excavate tumor markers secreted from blood or urine. It is necessary to analyze the biomarkers by a different approach and to diagnose liver diseases including liver cancer.

Although many tumor markers have been developed from blood, tissues, and excreta due to the above problems, many types of tumor markers may be increased or detected even without cancer. Therefore, the method of diagnosing cancer using these diagnostic markers is merely an incidental means, and is not itself used as an independent diagnostic tool.

On the other hand, the immune system is constructed as a unique system that distinguishes between self and non-self in the early stage of the generation of an individual. As a result, only the foreign antigen exposed to the immune system in the normal state of the individual, It develops to induce the humoral immune response and the cellular immune response. However, in the case of certain diseases, it has been confirmed that an antibody is also produced against self-antigen (self-antigen). In such a case, the expression site of the antigen is different from that of normal, Or other features appear to be different from normal individuals. It has been reported since the 1970s that autoimmune antibodies against cancer cell-derived antigens are produced in association with abnormal cancer cell growth even in the case of cancer, and these antigens are called tumor-associated antigens (TAA) . HER-2 / neu oncoprotein is known to induce autoimmune antibody as a receptor protein present in the cell membrane, and p53, a protein having cancer cell growth inhibitory function, is also known to be an autoimmune Lt; / RTI > antibody. In addition, cyclin B1 and CENP-F (centromere protein F), which are related to cell proliferation, and onconeurological proteins Hu and Yo are known to induce autoimmune antibodies. These results suggest that there will be more autoimmune antibodies against more cancer-associated antigens, and attempts to massively screen cancer-related autoimmune antibodies are continuing.

In addition, it has been suggested that cancer detection, which detects different autoimmune antibodies at once, may be much more effective than single autoimmune antibody. Therefore, studies on multiple detection of autoimmune antibodies have been suggested as important tasks. For example, it has been reported that by detecting autoimmune antibodies against ASB-9, SERAC1, and RELT, it is possible to diagnose breast cancer with 100% specificity and sensitivity of 80% (L. Zhong et al. Breast Cancer Res 10). In addition, the detection of autoimmune antibodies against PIM1, MAPKAPK3, and ACVR2B was able to diagnose colon cancer with a specificity of 74% and sensitivity of 83% (Babel et al. Mol Cell Proteomics 8: 10 (2009) 2382-2395). As such, cancer diagnosis using cancer-derived autoimmune antibodies has shown high efficacy, and new discoveries of cancer-derived autoimmune antibodies have become an important field of cancer biomarker derivation.

In order to identify an autoimmune antibody, SEREX (serological analysis of recombinant cDNA expression libraries of human tumors with autologous serum) technique for analyzing the blood reactivity of a cancer patient with a cancer expressing protein expression library has been conventionally used Respectively. However, this method has limitations in expressing the cancer cell-derived protein to have the maximum diversity at the library level, and since the final product of the protein undergoes posttranslational modification (PTM) even after transcription, Unrecognized protein expression libraries have obvious limitations that are insufficient for autoimmune antigen detection.

Recently, autoimmune antibody discovery studies based on proteomics techniques are under way as an alternative. In the proteomics technique, the protein derived from cancer cells is spread by two-dimensional electrophoresis (2D-PAGE), the protein spot showing the reactivity is confirmed by using the plasma of the cancer patient as the autoimmune antibody sample, It is also referred to as SERPA (serological proteome analysis). In addition, affinity chromatography resin, conjugated with antibodies isolated from patient blood, was prepared, and then cancer cell proteins were injected thereinto and MAPPING (Multiple Affinity Protein profiling techniques have also been used. In addition, it is possible to find an autoimmune antibody by dividing a cancer cell lysate into several thousands of fractions to prepare a protein chip and examining the reactivity of the patient's blood with the protein chip.

These various proteomic analytical techniques have the advantage of being able to directly confirm the antibody reactivity to the cancer cell-derived proteins which maintains the post-transcriptional modification (PTM) characteristics, so that it is possible to identify several autoimmune antibodies However, there are still other limitations. First, it is a quantitative problem of antibodies. If the subject to be analyzed is a mixture of two or more substances, the analysis of the amount of the substance is preferred, and the rest is likely to be excluded from the analytical range. The patient serum is a mixture of a large number of antibodies, The level of analytical ability can be determined by the quantitative difference of the antigen and the affinity for the antigen, and thus the analysis of the desired autoimmune antibody may not be possible. Second, because the autoimmune antibody sample to be analyzed is dependent on the patient, there is a limit to systematically analyzing the production of autoimmune antibodies according to the cancer onset condition, and it is difficult to collect an excessive amount of patient blood for the experiment. There is a limit in itself. Finally, it is a matter of preservation of the epitope where the antibody reacts. According to the known immunological knowledge, the antibody reaction site can be divided into two types, a protein sequence-dependent antigenic determinant (sequential epitope) and a protein structure-dependent antigenic conformational epitope to be. When an antibody against a specific antigen is induced in vivo, the state of the antigen that firstly contacts the immune cell antibody is reflected. This means that the antigen-antibody reaction proceeds in a solution state and the antigen protein remains dissolved in the blood It means that you have. Therefore, even in the case of an antigen-antibody reaction test conducted in vitro, it is preferable to conduct an antigen-antibody reaction test because it reflects the original binding state. However, in the case of the SERPA assay described above, two-dimensional electrophoresis, that is, the protein to be analyzed is denatured using SDS and urea, and the reaction with the antibody is examined in the unfolded state. Therefore, it is detectable when the antigenic determinant site is protein sequence dependent, but there is a limit that can not be detected when it is structure dependent.

As described above, the autoimmune antibody studies that can be used as the cancer-related markers reported in the past have a limited diagnostic effect despite the proposed application, and there is a limit to utilize the autoimmune antibody as a biomarker for cancer diagnosis. In addition, there are limitations in carrying out the method which does not include counts in many cases or requires excessive experimentation, so that autoimmune antibody markers for diagnosing liver cancer can not be found.

As a result of intensive efforts to develop an autoimmune antibody for the diagnosis of liver cancer, the present inventors have developed a method for identifying an effective cancer-associated autoimmune antibody in a liver cancer model mouse, and found that an autoimmune antibody specifically binding to ATIC Respectively. In addition, an ELISA method for detecting an autoimmune antibody was designed by screening a phage expressing an antigenic region (epitope) that specifically reacts with a peptide library, and it was confirmed that human liver cancer can be diagnosed using the ELISA method, thereby completing the present invention .

It is an object of the present invention to provide an autoimmune antibody that specifically binds to ATIC or a fragment comprising the antigen binding site thereof.

Another object of the present invention is to provide an antigen fragment which specifically binds to the autoimmune antibody of the present invention.

Another object of the present invention is to provide a composition for diagnosing liver cancer comprising an agent for measuring an expression level of an autoimmune antibody or a fragment containing an antigen binding portion thereof of the present invention.

It is still another object of the present invention to provide a hybridoma cell line producing the autoimmune antibody of the present invention.

It is still another object of the present invention to provide a kit for the diagnosis of liver cancer comprising the composition of the present invention.

It is still another object of the present invention to provide a method for providing information for diagnosis of liver cancer using the composition of the present invention.

It is another object of the present invention to provide a screening method for a therapeutic agent for liver cancer using the autoimmune antibody of the present invention.

In one aspect for accomplishing the above object, the present invention provides an autoimmune antibody that specifically binds to ATIC or a fragment comprising an antigen binding site thereof.

The term "ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase / IMP cyclohydrolase)" in the present invention is a bi-functional protein that catalyzes the final two step reaction of purine de novo biosynthesis. The biosynthesis of purine and pyrimidine is a nucleotide producing reaction necessary for the synthesis of DNA and RNA. It is rarely used in normal cells, but it acts actively on cells that divide rapidly as cancer cells. However, there are few studies related to ATIC and cancer outbreaks. In the present invention, anti-ATIC autoimmune antibodies were identified in the blood of patients with liver cancer for the first time, and a novel epitope structure responsive thereto was used to diagnose liver cancer .

The term "autoantibody " in the present invention means an antibody that specifically reacts with the body composition of the subject, and is also referred to as an autoantibody or autoantibody. Generally, an individual does not produce an antibody because it does not cause an immune response to the nature of its inherent identity. However, in certain cases, the substance itself may be recognized as an antigen to generate an antibody. The phenomenon that appears as a disease is autoimmune disease such as systemic lupus erythematosus and rheumatoid arthritis, ≪ / RTI > The autoimmune antibody of the present invention refers to an antibody that is produced against a cancer cell-derived antigen which is accompanied by a cancer cell growth that is not normal, unlike the above. Specifically, the antibody is generated against cancer- Anti-AIC autoimmune antibody "or" autoimmune antibody that specifically binds to ATIC ".

In one embodiment of the present invention, autoimmune antibodies obtained from hybridoma clones derived from liver cancer model mice were identified as "XC154 autoimmune antibody" or "TAB-XC154 autoimmune antibody" . As a result of identifying the specific reactive antigen protein for the antibody, it was confirmed that the antibody was finally ATIC having a molecular weight of 64.5 KDa, confirming that the autoimmune antibody was an anti-AIC autoimmune antibody (FIGS. 9 and 10)

In the present invention, the term "antigen-binding site (paratope)" means a site of an antibody binding to an antigen. The antigen binding site is a small region of the Fv region of the antibody and includes a heavy chain and a light chain portion of the antibody and binds to an antigenic determinant (epitope) of the antigen.

In the present invention, the fragment comprising the autoimmune antibody or its antigen binding site may preferably recognize the amino acid sequence selected from the group consisting of SEQ ID NOS: 17 to 20, more preferably the amino acid sequence shown in SEQ ID NO: 17 Lt; / RTI >

In one embodiment of the present invention, XC154 antibody-specific reactive antigens were screened from a peptide expression library to identify specific antigenic determinant sites of the autoimmune antibody. As a result, CLPSWFHRC (Cys-Leu-Pro-Ser- (SEQ ID NO: 17), CAPSWLHRC (Cys-Ala-Pro-Ser-Trp-Leu-His- Arg-Cys; SEQ ID NO: 18), CSPSGLFSC Leu-Phe-Ser-Cys; SEQ ID NO: 19) and CTPSWFHRC (Cys-Thr-Pro-Ser-Trp-Phe-His- Arg-Cys; SEQ ID NO: 20) (Cys-Leu-Pro-Ser-Trp-Phe-His-Arg-Cys; SEQ ID NO: 17) was found to be most reactive with the amino acid sequence of CLPSWFHRC .

In general, one antibody molecule has two heavy chains and two light chains, each heavy and light chain containing a variable region at its N-terminus. Each variable region consists of three complementarity determining regions (CDRs) and four framework regions (FRs), where the complementarity determining regions determine the antigen binding specificity of the antibody, Lt; / RTI > sequences that are relatively short. Preferably, the autoimmune antibody of the present invention comprises, as part of the heavy chain variable region, an autoimmune antibody consisting of the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 4 or the CDR3 sequence of SEQ ID NO: 5, And may be an autoimmune antibody or fragment thereof including all of the sequences of CDR1, CDR2 and CDR3. In addition, the autoimmune antibody of the present invention may comprise, as part of the light chain variable region, an autoimmune antibody consisting of the CDR1 sequence of SEQ ID NO: 6, the CDR2 sequence of SEQ ID NO: 7 or the CDR3 sequence of SEQ ID NO: And may be an autoimmune antibody or fragment thereof including all of the sequences of CDR1, CDR2 and CDR3. Further, it is obvious that the nucleic acid sequence encoding the above sequence is also included in the invention.

The autoimmune antibody of the present invention may more preferably be an autoimmune antibody consisting of the amino acid sequence of SEQ ID NO: 1 as the heavy chain variable region sequence or a fragment containing the antigen binding portion thereof and may be a light chain variable region sequence of SEQ ID NO: 2 An autoimmune antibody consisting of an amino acid sequence or a fragment comprising an antigen binding site thereof. Further, the heavy chain and the light chain may be used individually or together according to purposes, and a plurality of CDR sequences and light and heavy chains can be freely combined according to a conventional genetic engineering method as desired by a person skilled in the art.

In one embodiment of the present invention, the complementarity determining region (CDR) sequence was analyzed to investigate the characteristics of the autoimmune antibody. As a result, it was found that the autoimmune antibody of the present invention has the heavy chain CDR1 of SEQ ID NO: 3; A heavy chain CDR2 as set forth in SEQ ID NO: 4; And a heavy chain variable region comprising heavy chain CDR3 as set forth in SEQ ID NO: 5 and light chain CDR1 as set forth in SEQ ID NO: 6; A light chain CDR2 as set forth in SEQ ID NO: 7; And a light chain variable region comprising the light chain CDR3 represented by SEQ ID NO: 8, and was confirmed to be an autoimmune antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2 ).

The autoimmune antibody of the present invention comprises a heavy chain comprising two full-length or polynucleotides encoding a fragment having immunological activity of an antibody molecule capable of achieving antibody-antigen binding. In addition, the antibody of the present invention comprises a light chain comprising two full-length or polynucleotides encoding a fragment having an immunological activity of an antibody molecule capable of achieving antibody-antigen binding. Examples of antibody fragments include: (i) a variable region (V _L ) of a light chain and a variable region (V _H ) of a heavy chain and a constant region (V _H ) of an immunoglobulin A Fab fragment consisting of a region C _L and a first constant region C _H1 of the heavy chain; (Ii) an Fd fragment consisting of the V _H and C _H1 domains; (Iii) an Fv fragment consisting of the V _L and V _H domains of a monoclonal antibody; (Iv) a dAb fragment consisting of the V _H domain; (V) an isolated CDR region; (Vi) a F (ab ') 2 fragment that is a divalent fragment comprising two linked Fab fragments; (V) single chain Fv molecules (scFv) joined by a peptide linker that binds the V _H and V _L domains to form an antigen binding site; (Ⅷ) specific single chain Fv dimers and diabodies which are multivalent or multispecific fragments produced by (ⅸ) gene fusion, and the like.

The fragment comprising the autoimmune antibody or antigen-binding portion thereof of the present invention may preferably be a fragment comprising an autoimmune antibody or antigen-binding portion thereof produced by a hybridoma cell line of Accession No. KCTC 11873BP.

In another embodiment, the present invention provides an antigen fragment that specifically binds to an autoimmune antibody that specifically binds to the ATIC.

In the present invention, "an antigen specifically binding to an autoimmune antibody that specifically binds to ATIC" includes all the proteins capable of binding specifically to the autoimmune antibody, and is not limited to a specific protein or polypeptide. Preferably, the antigen may comprise all or a portion thereof, or a variant thereof, as long as it can be recognized by an anti-ATIC autoimmune antibody. The antigen may be composed of at least 9 amino acids, preferably at least 12 amino acids.

The antigen may be an antigenic determinant site sequence that can be recognized by the autoimmune antibody marker of the present invention. If the sequence is a sequence recognizable by the autoimmune antibody of the present invention, the sequence is not limited to the size or type of the sequence, and preferably the sequence may be a polypeptide sequence having cysteine at the end of seven amino acids. The sequence in which cysteine is present at the end of these seven amino acids is CLPSWFHRC (Cys-Leu-Pro-Ser-Trp-Phe-His-Arg-Cys; SEQ ID NO: 17), CAPSWLHRC -His-Arg-Cys; SEQ ID NO: 18), CSPSGLFSC (Cys-Ser-Pro-Ser-Gly-Leu-Phe- Ser-Cys; SEQ ID NO: 19) and CTPSWFHRC -His-Arg-Cys; SEQ ID NO: 20), and the presence of the cysteine may form a cyclic structure, and recognition by an autoimmune antibody may be more effective. However, the above examples do not limit the kinds of sequences that can be recognized by the autoimmune antibody of the present invention.

In another aspect, the present invention provides a composition for diagnosing liver cancer comprising an agent for measuring the expression level of the autoimmune antibody or a fragment containing the antigen binding site thereof.

In the present invention, the term "diagnosis" is intended to identify the presence or characteristic of a pathological condition. For the purpose of the present invention, the term " diagnosis " And so on. Preferably, when an autoimmune antibody that specifically binds to the ATIC of the present invention is used, the level of ATIC expression can be confirmed from a sample of a suspected individual of liver cancer, thereby determining whether or not the individual has a good prognosis Can be predicted.

In the present invention, the term "sample" refers to a group consisting of whole blood, serum, blood, plasma, saliva, urine, sputum, lymphatic fluid, cerebrospinal fluid and intercellular fluid in which the expression level of anti-ATIC autoimmune antibody, But it is not limited thereto.

In the present invention, the term "diagnostic marker, diagnostic marker, marker for diagnosis, or diagnosis marker" is a substance capable of distinguishing cancer cells from normal cells, (Such as mRNA), lipids, glycolipids, glycoproteins, or organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that exhibit an increase or decrease in cells with poor cancer . For the purpose of the present invention, the hepatocarcinoma diagnostic marker of the present invention is capable of specifically expressing a high level of expression in whole blood, blood, serum, plasma or the like of an individual showing liver cancer compared to whole blood, blood, serum or plasma of a normal liver RTI ID = 0.0 > anti-ATIC < / RTI > autoimmune antibody.

In the present invention, the term "liver cancer" generally refers to cancer originating from hepatocytes. Hepatocellular carcinoma (HCC) is a metastatic liver cancer that develops in the liver from primary liver cancer occurring in the liver and cancer occurring in other tissues. Most of the causes are unclear, but liver cirrhosis is often present, and hepatocellular carcinoma is found to occur in patients with cirrhosis, chronic active hepatitis B, or hepatitis B carriers. The present inventors confirmed that when the autoantibody marker of the present invention is used, diagnosis can be made with high sensitivity and specificity for the incidence of liver cancer from the individual.

In the present invention, an agent for measuring the expression level of an autoimmune antibody or a fragment containing an antigen-binding site thereof specifically binding to ATIC is expressed in an increased expression in whole blood, serum, plasma, lymph, Quot; means a molecule that can be used for the detection of a marker by identifying the level of expression of an anti-AIC autoimmune antibody or a fragment comprising the antigen binding site thereof, which is a marker that is a marker that specifically binds to the autoimmune antibody, Peptide. The polypeptide specifically binding to the autoimmune antibody is CLPSWFHRC (Cys-Leu-Pro-Ser-Trp-Phe-His-Arg-Cys; SEQ ID NO: 17), CAPSWLHRC Leu-His-Arg-Cys; SEQ ID NO: 18), CSPSGLFSC (Cys-Ser-Pro-Ser- Gly- Phe-His-Arg-Cys; SEQ ID NO: 20). Examples of analytical methods for this include Western blot, ELISA, radioimmunoassay, radial immunodiffusion, Oucherotin immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunization Precipitation assays, complement fixation assays, FACS and protein chips, but are not limited thereto. Antibody-antibody complex formation in the normal control group can be compared with the amount of antigen-antibody complex formation in the liver cancer or suspected individual through the above-described analysis methods. Thus, Diagnosis can be made.

A method for diagnosing liver cancer can be achieved through an antibody-antigen reaction between an anti-ATIC autoimmune antibody of the present invention and an antigen specifically binding thereto. In the present invention, the term "antigen-antibody complex" Refers to a combination of an immune antibody and a specific antigen, and the formation amount of an antigen-antibody complex can be quantitatively measured through the size of a signal of a detection label.

Such detection labels may be selected from the group consisting of enzymes, mimetics, ligands, emitters, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. Examples of enzymes that can be used when the enzyme is used as a detection label include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetyl Cholinesterase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate, Beta-decatase, beta-lactamase, and the like. Examples of the fluorescent material include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluorescamine and the like. Examples of ligands include, but are not limited to, biotin derivatives and the like. Examples of luminescent substances include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Examples of microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Les Examples of redox molecules are ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, 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. Examples of 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, It is not limited.

The term "peptide library" in the present invention refers to a peptide library of several hundred to several thousands of peptides that are artificially varied in amino acid sequence using combinatorial chemistry. Peptide library technology is a technology that can develop potential biologically active preliminary new drugs by searching for peptides having amino acid sequences of useful functions using peptide libraries. As a typical example, PS-SPCL (Positional-Scanning Synthetic Peptide Combinatorial Library) One-Bead-One-Peptide Library (OBOPL), and the like. However, the types of peptide libraries usable in the present invention are not limited by the above examples. Specifically, PS-SPCL was prepared by fixing 19 amino acids except cysteine and connecting 19 amino acid mixtures to the remaining positions. The mixture was composed of 114 pools of 6 residues and 114 samples were screened Is a technology that can search 47 million peptides at a time. Alternatively, the OBOPL library can be used to probe the attached sequences by allowing a resin-specific sequence (eg, a pentapeptide) to be attached to the polystyrene as a substrate, using polyethylene glycol (PEG) as a substrate. Preferably, the method for identifying an epitope sequence for an autoimmune antibody of the present invention includes the above-described identification method, but an epitope identification method usable in the art can be used without limitation. In order to identify the epitope sequence binding to the autoimmune antibody of the present invention, the present inventors used a phage display cyclic peptide library system in which a cysteine exists at the end of the sequence of seven amino acids to form a cyclic structure, -ATIC autoimmune antibody, while only the group of the selected phages having good reactivity with the anti-ATIC autoimmune antibody of the present invention as the antibody of the present invention is purified and treated with the coating antigen, These amino acid sequences were confirmed by confirming reactivity with the binding immunogens (Table 1).

As a result of studies on autoimmune antibodies reported in the past, most of them are expressed in normal cells except oncofetal proteins. In this case, as shown in the results of autoimmune antibody studies on GRP78, The major epitope is limited to one epitope. Therefore, detection of an autoimmune antibody is sufficient to use only a specific antigenic determinant site. As a result of examining the specific antigens recognized by the various autoimmune antibodies obtained from mice, the characteristics of the respective antigen proteins are different from each other. As a result, substantially all of the antigen proteins are produced as a recombinant protein, It is very difficult to propose. In particular, when the protein is large in size, such as when the molecular weight is 200 KDa or more, it is not easy to prepare the recombinant protein in its entirety. Therefore, when the present inventors propose an autoimmune antibody multi-detection method composed of autoimmune antigens, a method of presenting antigens in the form of expressing only the antigenic site that specifically reacts to the antibody on a common carrier protein is considered Respectively.

In addition, in order to identify autoimmune antibodies, the possibility of post translational modification (PTM), in which the autoantigen becomes new antigenic, should be considered. Most autoantigens are expressed in normal cells, but in the case of autoantigens accompanied by tumorigenesis, they are released from the cells due to changes in the post-transcriptional protein transcription, resulting in the formation of autoimmune antibodies In this case, the antigen of the autoimmune antibody is identified, and then the PTM is analyzed and reproduced, and the recombinant protein is prepared as the recombinant protein. It is impossible to detect an antigenic region having a PTM characteristic.

In order to solve these problems, the present inventors have used the phage peptide library to determine an antigenic determinant site (epitope). In view of the fact that the number of antigenic determinants of an autoimmune antibody to a specific protein is limited to one or two, identification of a single epitope through the search for a peptide library and detection of an autoimmune antibody can be made sufficiently by the presentation of this single epitope , And as a result of this experiment, the authors came to identify an autoimmune antibody recognizing an epitope determined by a peptide library.

Antibody, small protein, or peptide expression library technology using phage display technology has the advantage of quickly and repetitively screening antibodies, proteins or peptides that bind to a particular target and is useful for eliciting therapeutic antibodies, proteins or peptides . Generally, phage display technologies include 1) inserting a random sequence of oligonucleotides into a gene site corresponding to the N-terminus of the phage coat protein pIII (or pIVI); 2) expressing a fusion protein of a portion of the native form of the coat protein and the polypeptide encoded by the oligonucleotide of the random sequence; 3) treating a receptor material capable of binding to the polypeptide encoded by the oligonucleotide; 4) eluting the peptide-phage particles bound to the receptor using a low pH or competitive competitive molecule; 5) amplifying the phage eluted by panning in the host cell; 6) repeating the method to obtain the desired amount; And 7) determining the sequence of the active peptide from the DNA sequence of the phage clones screened by panning.

Since the epitope crystallization through the search of the peptide library makes it possible to select a structure having the most reactivity with the antibody, the structure can be used to search for the structure of the antigen itself, as well as the structure of the protein after the transcription. In addition, the selected antigen peptide expression phage can be easily propagated, and thus it is very easy to implement a diagnostic method such as the design of a protein chip.

In another aspect, the present invention provides a hybridoma cell line producing the autoimmune antibody of the present invention.

The term "hybridoma" in the present invention refers to a cell made by artificially fusing two different kinds of cells, using a substance causing cell fusion, such as polyethylene glycol, or a virus of a certain kind, Refers to a fused cell. Hybridomas integrate different functions of different cells into a single cell. A typical example of a hybridoma is a lymphocyte. Especially hybrid myeloma cells and B cells, the precursor cells of antibody-producing cells among the lymphocytes in the spleen or lymph nodes, are used for the research and clinical use because they produce monoclonal antibodies. In addition, T cells that produce lymphocaine (physiologically active substance) and hybrid cells that are tumor cells thereof have been put to practical use. Hybridomas producing the autoimmune antibodies of the present invention can be appropriately modified by those skilled in the art using cells known in the art. In one embodiment of the present invention, B-cell hybridoma cells were prepared by secreting spleen cells of HBx transgenic mice which were confirmed to have liver cancer as B cell group, and fusing with mouse myeloma cells Sp2 / 0, The hybridoma cell line producing the -XC154 clone secretory antibody was selected and deposited on February 21, 2011 at the Korea Biotechnology Research Institute's Biological Resource Center, and received the accession number KCTC 11873BP. Preferably, the hybridoma cell line can be a cell strain having the accession number KCTC 11873BP.

In another aspect, the present invention provides a kit for the diagnosis of liver cancer comprising the composition of the present invention.

The kit for the diagnosis of liver cancer of the present invention can be used not only as an antibody recognizing a primer, a probe or optionally a marker for measuring an expression level of an anti-ATIC autoimmune antibody or a fragment containing the antigen binding site thereof as a marker for diagnosing liver cancer, One or more other component compositions, solutions, or devices suitable for use in the present invention may be included.

Also, in the present invention, the kit for measuring the expression level of the protein expressed by the coding of the gene encoding the diagnostic marker ATIC may be used for immunological detection of the antibody. The kit may include a substrate, a suitable buffer solution, a chromogenic enzyme, A secondary antibody, and a chromogenic substrate. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The chromogenic enzyme may be peroxidase, Alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline- ), Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

As an assay method for measuring the protein level in the kit, Western blotting, ELISA, radioimmunoassay, radioimmunoassay, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, But are not limited thereto. Preferably, the kit of the present invention may be a kit using an ELISA method. The kit may be an ELISA kit.

ELISA methods for measuring protein expression levels include direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, use of a labeled antibody that recognizes a capture antibody in a complex of antibodies recognizing an antigen attached to a solid support Indirect ELISA, direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of antibody and antigen attached to the solid support, reaction with another antibody recognizing the antigen in the complex of antibody and antigen attached to the solid support And indirect sandwich ELISA using labeled secondary antibodies that recognize this antibody. For example, an antibody may be attached to a solid support, reacted with a sample, labeled with an antibody that recognizes an antigen of an antigen-antibody complex, and then labeled with an antibody that recognizes the antigen of the antigen-antibody complex The secondary antibody can be detected and detected by a sandwich ELISA method in which the secondary antibody is enzymatically developed. It is possible to confirm the onset of liver cancer by checking the expression level of anti-ATIC autoimmune antibody which is a diagnostic marker. Preferably, when the amino acid sequence selected from the group consisting of SEQ ID NOS: 17 to 20 is used as a capturing element for capturing an anti-ATIC autoimmune antibody, more preferably, when the amino acid sequence shown in SEQ ID NO: 17 is used, Road liver cancer can be diagnosed.

In one embodiment of the present invention, an epitope sequence that reacts with the autoimmune antibody of the present invention was identified using a peptide phage library. The identified sequence was used to react with a primary antibody, an autoimmune antibody in the sample, Human IgGAM-HRP as a secondary antibody was reacted with the anti-human IgGAM-HRP to determine the formation and formation amount of the antigen-antibody complex. As a result, it was possible to observe a pattern clearly distinguishable from the serum of normal individuals and liver cancer individuals (FIG. In the case of detecting an anti-ATIC autoimmune antibody and diagnosing liver cancer using such a method, liver cancer can be diagnosed with high specificity and sensitivity. In a preferred embodiment of the present invention, an enzyme-linked immunosorbent assay (ELISA) , It was confirmed that the sensitivity and the specificity of the normal individuals and the liver cancer individuals can be distinguished and diagnosed at a sensitivity of about 87% and a specificity of about 88% (FIG. 7). In addition, as a result of conducting the same method on the serum of breast cancer patients, it was confirmed that the method of the present invention is specific to the diagnosis of liver cancer, since the serum of breast cancer patients is not distinguished from the normal (FIG. 8).

In the present invention, the term "sensitivity" refers to a rate at which a disease entity is positively detected, and when a diagnostic method is used, it is a criterion for how well the subject is actually found. In addition, the term "specificity" refers to the rate at which a normal individual is detected by speech, and is a criterion for how well individuals that do not belong to the disease are classified.

Western blots using one or more antibodies to the diagnostic marker may also be used. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the protein produced by the expression of the gene can be confirmed by confirming the amount of the generated antigen-antibody complex using the labeled antibody, thereby confirming whether or not the liver cancer has developed. In addition, tissue immuno staining using one or more antibodies against the marker can be performed. Tissues are harvested and fixed from hepatocarcinogenic or suspected individuals and paraffin format blocks are prepared by methods well known in the art. These fragments are made into a slice of several 탆 thick, attached to a glass slide, and then reacted with a fragment containing the antigen-binding site of the autoimmune antibody of the present invention by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope. In addition, a protein chip in which one or more antibodies against the marker are arranged at predetermined positions on a substrate and immobilized at high density can be used. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It is possible to confirm whether or not the liver cancer develops.

In another aspect, the present invention provides a method for diagnosing liver cancer, comprising the step of detecting a fragment containing an autoimmune antibody or an antigen-binding site thereof that specifically binds to ATIC using the composition of the present invention Provide a method of providing.

The method for providing information for the diagnosis of liver cancer according to the present invention is preferably a method comprising the steps of (a) detecting the level of expression of an autoimmune antibody or its fragment containing an antigen-binding site specifically binding to ATIC from a sample of a subject suspected of having liver cancer Measuring; And (b) comparing the expression level to an expression level of a fragment comprising an autoimmune antibody or antigen-binding portion thereof that specifically binds ATIC of a normal control sample.

In the present invention, the term "animal" includes, but is not limited to, a horse, a dog, a cat, a pig, a goat, a rabbit, a hamster, a monkey, a guinea pig, a rat, Snakes, sheep, cattle, fish and birds, preferably human.

In the present invention, the term "control group" refers to a sample derived from an individual in which the expression of an anti-ATIC autoimmune antibody is expressed at a level lower than that of a liver cancer-causing or suspected individual. It is a sample that is the standard for diagnosis of liver cancer by antibody reaction.

In the present invention, the term "sample" refers to a group consisting of whole blood, serum, blood, plasma, saliva, urine, sputum, lymphatic fluid, cerebrospinal fluid and intercellular fluid in which the expression level of anti-ATIC autoimmune antibody, But it is not limited thereto.

As a method for measuring the expression level of the protein in the present invention, Western blotting, ELISA, radioimmunoassay, radioimmunoassay, Ouchteroni immunodiffusion, But are not limited to, immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS and protein chip methods. Details of the method for measuring the expression level of the protein are as described above.

In another aspect, the present invention provides a screening method for a therapeutic agent for liver cancer using the autoimmune antibody of the present invention. Specifically, (a) measuring the level of expression of an autoimmune antibody specifically binding to ATIC or a fragment comprising an antigen-binding portion thereof; (b) administering a candidate substance for the treatment of liver cancer; And (c) confirming that the expression level of the fragment comprising the autoimmune antibody or its antigen binding site specifically binding to ATIC is reduced as compared to the step (a). do.

In the present invention, the step (a) is a step of measuring the expression level of an autoimmune antibody specifically binding to ATIC or a fragment containing an antigen-binding site thereof, and a method of measuring the normal expression level as described above Can be used without restrictions.

In the present invention, the steps (b) and (c) may include the steps of administering a candidate substance for the treatment of liver cancer and a step of administering the candidate substance for treatment of liver cancer, wherein the expression level of the fragment comprising an autoimmune antibody or antigen- Step is confirmed.

The term "candidate substance for treating liver cancer" in the present invention is a substance expected to treat liver cancer, and can be used without limitation as long as it is a substance expected to improve or improve liver cancer directly or indirectly. Or proteins, and the like. The screening method of the present invention confirms the expression level of the fragment containing the anti-ATIC autoimmune antibody or its antigen binding site before and after the administration of the candidate substance, and when the expression level is decreased compared to before the candidate substance administration, Candidate substances can be determined as therapeutic agents for liver cancer.

When the anti-AIC specific autoimmune antibody of the present invention is used as a marker for liver cancer, non-invasive biological samples such as blood, plasma, serum, and lymphatic fluid can be used to obtain sensitivity of about 87% About 88% of cases can diagnose the onset of liver cancer in a specific way. Further, the present invention can easily diagnose liver cancer using only the identified amino acid sequence without designing a complex reaction substance to identify the marker by identifying the sequence that reacts with the marker, and is effective in developing a kit for the diagnosis of liver cancer to be.

FIG. 1 shows a method for securing XC154 autoimmune antibody derived from liver cancer mouse and a method for diagnosing cancer using the same. The XC154 antibody was obtained by confirming the reactivity of autoimmune antibodies obtained from HBx liver cancer model B-cell hybridoma clones to liver cancer cells. HepG2 and Hepa1c1c7 cells were immobilized on the hepatocellular carcinoma cell line. After permeabilization of the cell membrane, autoimmune antibodies from mouse liver model mice were treated. Secondary antibodies with fluorescent labeling were confirmed by flow cytometric ananlysis. The phage that mimic the epitope corresponding to the XC154 antibody obtained by the panning method was selected using a phage display peptide library kit (Ph.D.-C7C Phage Display Peptide Library kit (New England Biolabs)) forming a cyclic structure And a method to identify the difference between the liver cancer patient and the normal person using the selected phage.
FIG. 2 shows the results of analysis of the complementarity determining region (CDR) of the heavy chain mutation region (V _H ) and the light chain mutation region (V _L ) of the XC154 antibody obtained as a CDR sequence of TAB-XC154 autoimmune antibody. CDNA was synthesized for total RNA (total RNA) extracted from XC154 antibody-producing B cell hybridoma, amplified by polymerase chain reaction (PCR) using primer for complementary site for complementation of heavy chain region and complementary site for light chain mutation region And then ligated to a pTOP Blunt V2 vector to prepare a recombinant plasmid. The recombinant plasmid was amplified in the host cell and extracted, and the corresponding site was sequenced. Protein sequences were identified from the analyzed nucleotide sequences and CDR sequences were determined based on Kabat CDR definitions.
Fig. 3 shows results of oily cell analysis (a) and intracellular staining (b) for confirming the reactivity of XC154 antibody against various cancer cells. The XC154 antibody reactivity to various cancer cells was analyzed by an oily cell assay after staining in cancer cells. As a result, it was confirmed that not only liver cancer cell lines such as HepG2 and SK-Hep1 but also other cancer cells showed high reactivity. In addition, the intracellular expression site of the antigen protein recognized by the XC154 antibody was confirmed by fluorescence staining. As a result, in the cancer cell hosts HepG2 and Hepa-1c1c7, the cytoplasm was concentrated to the cell membrane side, .
Figure 4 shows the results of Western blotting for identification of XC154 autoimmune antibody specific reactive antigen protein. Cell debris and serum-free cell culture of various types of cancer cell lines were quantified and developed on 10% SDS-PAGE in 50 ㎍ increments. Western blotting with XC154 antibody was performed. As a result, molecular weight of about 64 KDa was recognized (M, molecular weight marker; 1, Hepa1c1c7; 2, HepG2; 3, HT-29).
FIG. 5 shows the result of screening of the phage expressing XC154 antibody-specific reactive epitope by screening the phage circular peptide library with XC154 antibody as a result of screening of the TAB-XC154 antibody-specific reaction epitope. In FIG. 5 (a), panning using XC154 antibody was carried out five times, and four phages (XC154p1, XC154p2, XC154p4 and XC154p9) having different peptide sequences were selected. In FIG. 5 (b), the reactivity of the XC154 antibody to the XC154p1 phage was confirmed by ELISA. As a result, it was confirmed that the XC154p1 phage had the highest specificity for TAB-XC154 antibody.
Figure 6 shows competitive inhibition of the cellular response of the TAB-XC154 antibody by the XC154p1 epitope-expressing phage. XC154p1 phage screened to mimic the epitope structure of an antigen that binds to TAB-XC154 antibody competitively inhibits TAB-XC154 antibody-antigen binding. HepG2, a prostate cancer cell line, and LNcap / LN3, a proliferating cancer cell line, were treated with an XC154 antibody that had been previously reacted with XC154p1 phage after permeabilization of the cell membrane, and a secondary antibody with a fluorescent label was identified by the oily cell assay . As a result, it was confirmed that the XC154p1 phage almost completely inhibited the antibody response of the cell-expressing antigen, and thus it was confirmed that the phage expression epitope well mimics the antigenic site structure of the original antigen.
Figure 7 shows the results of the diagnosis of liver cancer using the XC154p1 epitope-expressing phage. TAB-XC154 antibody-specific reaction peptides XC154p1 as a coating antigen was assayed by ELISA for hepatocellular carcinoma and normal human serum. As a result, it was confirmed that the sensitivity of 86.96% and the specificity of 88.24% were able to distinguish patients with liver cancer from the cutoff value of 0.059.
Fig. 8 shows the results of the diagnosis of breast cancer using the XC154p1 epitope-expressing phage. When ELISA using XC154p1 XC154p1 as a coating antigen was used for the analysis of breast cancer patients and normal persons, the serum of breast cancer patients was not differentiated from the serum of normal persons, unlike the case of liver cancer.
Figure 9 shows the identification of the XC154 antibody specific reaction antigen protein. Figure 9a shows the purification of the XC154 antibody reactive antigen protein. The LNCap-LN3 cell disruption solution, in which TAB-XC154 antibody-reactive antigen was overexpressed, was prepared and fractionated with HitrapQ-sepharose, an anion exchange resin, to partially purify the corresponding antigen. Only the fractions in excess of the corresponding antigens were concentrated, developed on SDS-PAGE, extracted with XC154 antigen only, and subjected to in-gel digestion, followed by protein analysis using a jelly analysis method. Fig. 9 (b) shows the results of mass spectrometry of the XC154 antibody-specific reaction antigen protein. As a result of protein identification of XC154 antigen, ATIC was found to have the highest score. ATIC is involved in purine biosynthesis and is known as a protein that plays an important role in the proliferation of cancer cells. The calculated protein molecular weight is 64.5 KDa, similar to the molecular weight of XC154 antigen.
Figure 10 shows the validation of the identified XC154 antibody specific reaction antigen protein. Fig. 10A shows inhibition of ATIC expression using siRNA. SiRNA specific to ATIC was transfected into HepG2 cells, and after 48 hours, expression of the gene was confirmed by RT-PCR. As a result, it was confirmed that the expression was inhibited by 50% or more. 10 (b) shows the results of the oily cell analysis for confirming the XC154 antibody reaction after the inhibition of ATIC gene expression. As a result of confirming the reactivity of XC154 antibody against cells treated with siRNA in the same manner as above, it was confirmed that the antibody reaction was reduced by 20%. FIG. 10C shows the result of Western blotting for confirming the XC154 antibody reaction after suppression of ATIC gene expression. Western blotting was performed to confirm that the expression-inhibited protein was an XC154 antibody response protein. As a result, it was confirmed that XC154 antibody was not reacted with ATIC expression suppressed cells, and XC154 antigen was found to be ATIC.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Securing XC154 autoimmune antibody from HBx mouse

HBx transgenic mice, which have already been reported to produce similar types of hepatocellular carcinoma to human liver carcinomas, are used to obtain autoimmune antibodies that are produced in association with cancer development. B cell hybridoma cells were prepared by secreting spleen cells of individuals with hepatocellular carcinoma HBx transgenic mice in the breeding as B cell group and cell fusion with mouse myeloma cell Sp2 / 0. Cell fusion followed the general B cell hybridoma preparation. Fusion cells were firstly screened with HAT medium (hypoxanthine-aminopterin-thymidine medium), and only the cells that formed the clones were cultured separately. Among these, only the cells in which the cancer cell reactive antibody was detected in the cell culture medium were again selected and maintained.

Identification of the reactivity of the autoimmune antibody against cancer cells was carried out by intracellular staining of immobilized and permeabilized cancer cell lines followed by flow cytometric ananlysis analysis. The details are as follows. 70 ~ 80% confluency state of HepG2 or Remove the Hepa1c1c7 cells from the cell culture plate at a trypsin treatment, washed with PBS 2 ㅧ 10 ⁵ a Cytofix / Cytoperm solution (BD, Inc.) of 100 ㎕ per cell at 4 ℃ 20 bungan treatment To fix and permeate. After the reaction, 1 ml of Cytowash / Cytoperm solution (BD) was added, mixed well, and centrifuged at 1700 rpm for 5 minutes to obtain cell sediment. To the washed samples, 50 μl of primary antibody solution (B cell hybridoma cell culture solution or purified primary antibody solution) was added and reacted at 4 ° C for 40 minutes. After the reaction, cells were washed three times and treated with anti-mouse Igs-FITC (anti-mouse Igs-FITC (DaKo)) at 4 ° C for 40 minutes. After the reaction, the cells were washed three times, and the cells were precipitated in 300 μl of PBS and analyzed by FACScalibur (BD). The mean fluorescence values corresponding to the antibody responses were obtained and the responses were compared. In the control group without antibody reaction, 50 쨉 l of DMEM medium without primary antibody was added.

As a result, it was possible to confirm various auto-immune antibody-producing clones among B cell hybridoma cells. In the present invention, one of them was analyzed for a TAB-XC154 clone secretory antibody (FIG. 1). Thus, a hybridoma cell line producing the TAB-XC154 clone secretory antibody was deposited with the KRIBB Biological Resource Center (KCTC) on Feb. 21, 2011 and received the accession number KCTC 11873BP.

Example 2 Analysis of Complementary Determining Region (CDR) of XC154 Antibody and Purification of Antibody

After confirming that the XC154 antibody specifically recognized the antigen expressed in cancer cells, Complementary Determination Region (CDR) sequence of the antibody was analyzed in order to obtain information on the antigen specificity of the XC154 antibody. The detailed method is as follows.

About 10 ⁶ XC154 antibody-producing cells were collected and total RNA (total RNA) was extracted using an RNA extraction kit (Qiagen). CDNA was synthesized from 5 쨉 g of total RNA using a complementary DNA (cDNA) synthesis kit (Invitrogen), and 1 쨉 g of the synthesized cDNA and 5 'of mouse heavy chain constant region primer F 5' (SEQ ID NO: 21), R5'-GGA AGA TCT GAC ATT TGG GAA GGA CTG ACT CTC-3 '(SEQ ID NO: 22) and mouse light chain region primers 3 '(SEQ ID NO: 23), R 5'-GGT GCA TGC GGA TAC AGT TGG TGC AGC ATC-3' (SEQ ID NO: The CDR region of the heavy and light chains of the mouse was amplified by polymerase chain reaction (PCR) using SEQ ID NO: 24 and then ligated to the pTOP Blunt V2 vector using a TOPcloner Blunt kit (Enzynomics) . Recombinant plasmids were transformed into E. coli DH5α and plated on LB-restricted medium plates containing ampicilin and incubated for 15 hours at 37 ° C. One of the colonies formed on the plate was cultured in an LB liquid-restricted medium for 12 hours or longer, and the plasmid was extracted and the corresponding nucleotide sequence was analyzed. The protein sequence was determined from the analyzed nucleotide sequence, which was analyzed based on the Kabat CDR definition to determine the CDR of the XC154 antibody (FIG. 2).

For the analysis of the XC154 antibody specific reaction, a single-component purified autoimmune antibody is required. Therefore, in the present invention, a cell culture solution or an antibody-producing cell in which a large amount of XC154 antibody-producing clone was cultured was injected into mouse abdominal cavity to obtain ascites fluid and used for antibody purification. XC154 antibody was confirmed to be IgM as a result of isotyping, and thus MBP-agarose (Mannose-binding protein immobilized agarose, Pierce) or Protein L agarose for affinity chromatography for IgM purification agarose) was used. The purified antibodies were identified by comassie staining after SDS-PAGE and quantified by the Bradford method.

Example 3 Expression of XC154 Antibody-Specific Reactive Antigen in Various Cancer Cells

In order to confirm the reactivity of XC154 autoimmune antibody against various cancer cell lines, various cancer cells were stained intracellularly as in Example 1 and analyzed by flow cytometric ananlysis (FIG. 3). As a result, it was confirmed that expression of XC154 antibody reaction protein was overexpressed in liver cancer cell lines such as HepG2, SK-Hep-1, and cancer cells of various other species such as HeLa, LNcap-LN3 and A549.

In addition, intracellular staining was performed by confocal fluorescence microscopy to confirm the expression position in the cells, and staining was strongly observed at the cytoplasmic or cell membrane positions (Fig. 3).

Example 4: Identification of XC154 autoimmune antibody specific reaction antigen protein

Expression of XC154 antibody specific reaction antigen protein was confirmed by Western blotting in cancer cell lines. The cells to be analyzed were collected and dissolved in NP40 buffer (NP40 buffer: PBS containing 1.0% (v / v) NP40, protease inhibitor cocktail (Roche)) and used as a protein analysis sample. Protein quantification was performed by the Bradford method. The prepared protein samples were developed in 50 μg of SDS-PAGE under 10% reduced conditions and transferred to a PVDF membrane. After the transfer, the membrane was immersed in 5% (w / v) skim milk / TBS (Tris-buffered saline) for blocking and treated with the primary antibody. As the primary antibody, purified XC154 antibody was diluted in blocking solution at a concentration of 0.1 μg / ml. After primary antibody treatment, the cells were washed thoroughly with TBST (TBS containing 0.1% (v / v) tween-20) and treated with secondary antibody (anti-mouse IgGAM-HRP) followed by enhanced chemiluminescence To identify antibody response protein bands. As a result, the XC154 antibody specific reaction antigen protein in the HepG2 liver cancer cell line and the HT29 colon cancer cell was confirmed to have a molecular weight of about 64 KDa (FIG. 4).

Based on the above results, it was confirmed that the autoimmune antibody from liver cancer mouse recognizes a specific protein expressed in human liver cancer cells. Based on this finding, the antibody detection method can be derived by screening the antigenic determinant site of XC154 antibody from a random peptide expression library Respectively.

Example 5: Detection of XC154 antibody specific reaction peptide antigen expression phage

XC154 antibody-specific reaction The antigenic determinant is the most important factor in the construction of the detection method for determining whether an autoimmune antibody against the same antigenic determinant is present in human serum. XC154 antibody specific reaction peptide antigen was screened from a random peptide expression library in order to utilize only the antigenic determinant site without using the whole antigen protein as an antibody specific reaction site. The peptide expression library includes a phage display peptide library kit (Ph.D.-C7C Phage Display Peptide Library kit) in which seven amino acids are randomly expressed and cysteine residues are present at both ends to form a cyclic structure of seven amino acids England Biolabs) was used and the panning procedure followed the manufacturer's suggested method.

300 ng of XC154 antibody was mixed with 200 μl TBST solution containing 2 ㅧ 10 ¹¹ different peptide expressing virions and incubated at room temperature for 20 minutes and preblocked solution (0.1 M NaHCO ₃ , pH 8.6, 5 and 25 μl of protein L-agarose bead treated with 0.02% (w / v) NaN ₃ ), and allowed to react at room temperature for 15 minutes. Antibody-reaction phages were collected in the form of a precipitate by centrifugation in the form of antibody-virion-protein L agarose conjugate, washed several times with TBST, and eluted with a pH of 2.2 (0.2 M Glycine-HCl, pH 2.2, 1 mg / ml BSA). And quickly converted to neutral pH by the addition of 1 M Tris-HCl solution at pH 9.1. Some of the eluted phages were used to titrate the phage and the remainder were used to amplify the phage. The amplified phages were again panned in the manner described above.

As a result, a phage having four different peptide sequences was obtained after five rounds of panning on the XC154 antibody. In the panning process using the XC154 antibody, the number of phages amplified by the number of times up to the fourth was increased, and it was conceivable that the phages having the specific binding were amplified (FIG. 5A). 10 of the obtained phages were randomly selected and the sequence of the peptide region was determined by DNA sequencing and the amino acid sequence was determined (Table 1). The sequence of PSWFHR was confirmed repeatedly in the sequence of six antigenic monomers, and it could be assumed that it is important as the sequence of the antigenic monocyte.

In order to confirm the reactivity of the phages selected as specific binding to the XC154 antibody to the XC154 antibody, the phage was used as the coating antigen and the XC154 antibody as the primary antibody was subjected to ELISA. The details are as follows. As a result of peptide sequence determination, it was confirmed that phages (XC154p1, XC154p2, XC154p4, XC154p9) of different sequences were selected, and they were amplified and partially purified by PEG / NaCl solution treatment and used as an ELISA coating antigen. Purified phages were diluted by 10 ¹⁰ in 100 μl of the coating solution (0.1 M sodium bicarbonate buffer, pH 8.6) and added to each well of a 96-well Maxisorp ELISA plate. For antigen coating, the phage-added plates were stored at 4 ° C for at least 16 hours. After phage coating, 300 μl of skim milk solution (5% (w / v) skim milk / TBST) was added and reacted at room temperature for 1 hour to block the remaining portion after antigen coating. After blocking, the cells were washed twice with TBST (TBS containing 0.1% Tween-20), and 100 ng / 100 μl of XC154 antibody was added thereto, followed by reaction at room temperature for 90 minutes. After the reaction, the cells were washed 6 times with TBST and the secondary antibody anti-mouse IgGAM-HRP (Pierce) was diluted at a ratio of 1: 2500. The secondary antibody was also reacted at room temperature for 90 minutes and then washed with TBST for 6 times. Then, TMB solution (Pierce) was used as a substrate for HRP, and a color reaction was performed. The absorbance was measured at 450 nm to quantitate the antigen-antibody reaction. As a result, it was confirmed that XC154p1 among the four phages used as an antigen showed the highest reactivity (Fig. 5B).

Example 6: Competitive inhibition of liver cancer cell response of XC154 antibody using XC154p1 epitope expression phage

In order to confirm whether the antibody-specific phage were sufficiently replicating the antigenic determinant site (epitope) of the cancer cell-expressing antigen protein, when the reaction of the XC154 antibody with the cell-expressed antigen protein was determined by oily cell analysis, the XC154 antibody- To confirm that the reaction was inhibited competitively. The details of the oily cell measurement method are the same as those described in Example 1. The primary antibodies were prepared by XC154 antibody alone and XC154p1 antibody for 1 hour. At this time, 100 ng of antibody was used and 10 ¹¹ or 10 ¹² phages were used. As a control, M13 phage (Eph), which does not express the antigenic peptide, was used and the reaction was confirmed in HepG2 and LnCap-LN3 cells.

6, it can be seen that the XC154p1 phage suppresses the response of the XC154 antibody against HepG2 or LnCap-LN3 cells by more than 90% under the above conditions. These results demonstrate that the XC154p1 phage mimics the epitope region of the XC154 antibody-specific reactive antigen protein.

Example 7: Diagnosis of liver cancer using XC154p1 epitope expression phage

In general, the site (epitope) where protein antigens stimulate the immune system to induce an antibody response is limited to specific sites within 20 amino acids of the total protein structure. It is well known that the characteristics of these epitope regions are similar to those of human, mouse, Based on these facts, it is expected that the autoimmune antibody response epitope obtained from liver cancer model mouse will have the same effect on human body. Therefore, it was confirmed that XC154p1 phage with XC154 antibody specificity can be detected in the detection of human autoimmune antibody, thereby detecting an autoimmune antibody in human serum. The details are as follows.

100 ng of anti-M13 phage antibody per each well of the ELISA plate was diluted in 100 μl of 0.1 M sodium bicarbonate buffer, pH 8.6 solution, and coated at 4 ° C for 16 hours. After coating, the excess antibody was removed and blocking was performed by adding 300 μl of protein-free blocking buffer (Pierce) to each well. After blocking, the cells were washed twice with TBST and treated with 10 ¹⁰ XC154p1 phages per well diluted in 100 mu l of protein-free blocking buffer. After treatment, the cells were washed 6 times with TBST, and hepatocellular carcinoma patients and normal human serum were treated with primary antibody. The primary antibody reaction was carried out at room temperature for 2 hours. After the reaction, the remaining antibodies were washed 6 times with TBST. As a secondary antibody, anti-human IgGAM-HRP (anti-human IgGAM-HRP, Pierce) was diluted 100: 1 in a protein-free blocking solution at a ratio of 1: 5000 and reacted at room temperature for 90 minutes. After the reaction, the cells were rinsed with TBST for 6 times, and 100 μl of TMB solution was added thereto, followed by HRP reaction, and the absorbance at 450 nm was measured. The ELISA for human serum using XC154p1 was repeated three times or more to confirm reproducibility, and the most representative results thereof are shown in FIG.

As can be seen from FIG. 7, the XC154p1 phage was able to differentiate the serum of normal and liver cancer patients with a sensitivity of 86.96% and a specificity of 88.24%. These results suggest that the human serum ELISA method using the XC154p1 phage, which is the XC154 antibody antigenic region mimic of the present invention, can be utilized as a diagnostic method for liver cancer.

Example 8: Diagnosis of breast cancer using XC154p1 epitope expression phage

In order to confirm the diagnosis of breast cancer patients using the XC154p1 phage, the ELISA method prepared by the same method as above was also carried out on the serum of breast cancer patients.

The XC154 antibody was also highly reactive with autoimmune antibodies obtained from liver cancer model mice or other types of cancer cell lines, so that a diagnosis of breast cancer could be expected. However, as shown in FIG. 8, in the case of breast cancer patients, And the results were not different.

In conclusion, the XC154p1 phage antigen, which simulates the antigenic site of XC154 autoimmune antibody, could be used as an ELISA diagnostic method for the diagnosis of liver cancer.

Example 9: Identification of the protein recognized by the XC154 antibody

These results suggest that XC154 autoimmune antibody is a significant biomarker in liver cancer. To further elucidate the significance of XC154 autoimmune antibody in liver cancer, identification of antigen protein and its role in cancer development was required. Accordingly, the present inventors performed identification of the XC154 antibody-specific reaction antigen protein. The details are as follows.

XC154 antibody-specific reaction To identify the antigen protein, the LN-Cap-LN3 cell disruption solution (using RIPA buffer), which has high expression of the corresponding antigen protein, was fractionated into HiTrap-Q (GE healthcare) column. From the Western blotting results for each fraction, fractions containing the XC154 antibody specific antigen protein were selected and the fractions were concentrated by acetone precipitation. The concentrated antigen protein solution was separated on an 8-10% SDS-PAGE gel, some of which was used to confirm the presence of XC154 antigen by Western blotting, the remaining protein band was identified by coomassie staining, and then the XC154 antibody reaction site The corresponding protein bands were excised and subjected to in-gel digestion reaction using trypsin protease (Fig. 9 (a)). In addition, information on the protein sequence was obtained by mass spectrometry on peptide fractions extracted after in-gel digestion, and it was confirmed from the result that the XC154 antibody-reactive antigen was 5-aminoimidazole-4-carboxamidibonucleotide formyl-transferase / IMPcyclohydrolase 9, b).

In order to verify the results of protein identification by mass spectrometry, we confirmed the decrease of XC154 antibody response in cells inhibiting ATIC expression using siRNA. The details are as follows. The siRNAs that inhibit the expression of ATIC were siRNAs supplied by Bioneer Co., and were injected into HepG2 cells using lipofectamine 2000 (Lipofectamine 2000, Invitrogen). After 48 hours of siRNA treatment, the cells were harvested and total RNA was extracted and cDNA was synthesized using 5 의 of RNA as a template. The synthesized cDNA was used as a template and polymerase chain reaction was performed with primers having specificity for ATIC gene. The result was analyzed on 1% agarose gel (FIG. 10A). As a result, it was confirmed that the expression of ATIC was inhibited by 50% or more. Flow cytometric analysis of the XC154 antibody against cells confirmed to be inhibited by the expression of ATIC by RT-PCR was carried out in the same manner as described above. As a result, it was confirmed that the XC154 antibody-specific reaction was decreased in the state where the expression of ATIC was suppressed (Fig. 10 (b)). This tendency was confirmed in Western blotting results. Western blotting after treatment with siRNA against ATIC resulted in a significant decrease in the amount of XC154 antibody reactive antigen protein (Fig. 10 (c)). These results confirmed that XC154 antibody specific antigen was ATIC.

Korea Biotechnology Research Institute KCTC11873BP 20110221

<110> Korea Research Institute of Bioscience and Biotechnology <120> A marker comprising anti-ATIC autoantibodies and a composition          a cancer of the liver <130> PA110234KR <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> a heavy chain variable region <400> 1 Gly Xaa Lys Leu Gln Gly Ser Gly Ala Gly Leu Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Tyr              20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Gly Trp Ile          35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Ile Asn Tyr Asn Gly Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Arg Thr Ala Tyr  65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Gly Asp Ser Ala Val Tyr Phe Cys                  85 90 95 Ala Arg Gly Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala             100 105 110 Gly Ser Gln Ser Phe Pro Asn Val         115 120 <210> 2 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> a light chain variable region <400> 2 Xaa Ile Val Leu Xaa Xaa Xaa Thr Ala Ile Met Ser Ala Ser Pro Gly   1 5 10 15 Gly Lys Val Thr Met Thr Cys Ser Ala Ser Ser Val Asn Asn Met              20 25 30 Tyr Trp Tyr Gln Gln Lys Thr Gly Ser Ser Pro Arg Leu Leu Ile Tyr          35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Val Val Arg Phe Ser Gly Asn      50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Gly Ala Gly  65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Phe Thr                  85 90 95 Phe Gly Gly Gly Thr Lys Leu Gly Leu Lys Arg Ala Asp Ala Ala Pro             100 105 110 Thr Val Ser         115 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of a heavy chain variable region <400> 3 Gly Tyr Ala Phe Ser Asn Tyr Trp Met Asn   1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of a heavy chain variable region <400> 4 Gln Ile Tyr Pro Gly Asp Gly Asp Ile Asn Tyr Asn Gly Lys Phe Lys   1 5 10 15 Gly     <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of a heavy chain variable region <400> 5 Gly Thr Tyr   One <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of a light chain variable region <400> 6 Ser Ala Ser Ser Val Asn Asn Met Tyr   1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of a light chain variable region <400> 7 Asp Thr Ser Asn Leu Ala Ser   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of a light chain variable region <400> 8 Gln Gln Trp Ser Ser Tyr Pro Phe Thr   1 5 <210> 9 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> a heavy chain variable region <400> 9 gaggtkaagc tgcaggagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatt 60 tcctgcaaaa cttctggcta cgcattcagt aactactgga tgaattgggt gaagcagaga 120 cctggaaagg gtcttgagtg gattggacag atttatcctg gagatggtga tattaattat 180 aatggaaagt tcaagggcaa ggccacactg actgcagaca aatcctccag aacagcctac 240 atgcaactca gcagtctgac ctctgaggac tctgcggtct atttctgtgc aagagggact 300 tattggggcc aagggactct ggtcactgtc tctgcagaga gtcagtcctt cccaaatgtc 360                                                                          360 <210> 10 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> a light chain variable region <400> 10 gayattgtgc tcacmcarwc tacagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaaat aacatgtact ggtatcagca gaagacagga 120 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgttcgt 180 ttcagtggca atgggtctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccaacagtgg agtagttatc cattcacgtt cggagggggg 300 accaagctag aactaaaacg ggctgatgct gcaccaactg tatcc 345 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CDR1 of a heavy chain variable region <400> 11 ggctacgcat tcagtaacta ctggatgaat 30 <210> 12 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> CDR2 of a heavy chain variable region <400> 12 cagatttatc ctggagatgg tgatattaat tataatggaa agttcaaggg c 51 <210> 13 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> CDR3 of a heavy chain variable region <400> 13 gggacttat 9 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CDR1 of a light chain variable region <400> 14 agtgccagct caagtgtaaa taacatgtac 30 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CDR2 of a light chain variable region <400> 15 gacacatcca acctggcttc t 21 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CDR3 of a light chain variable region <400> 16 caacagtgga gtagttatcc attcacg 27 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> epitope of XC154p1 <400> 17 Cys Leu Pro Ser Trp Phe His Arg Cys   1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> epitope of XC154p2 <400> 18 Cys Ala Pro Ser Trp Leu His Arg Cys   1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> epitope of XC154p4 <400> 19 Cys Ser Pro Ser Gly Leu Phe Ser Cys   1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> epitope of XC154p9 <400> 20 Cys Thr Pro Ser Trp Phe His Arg Cys   1 5 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> heavy chain constant region forward primer <400> 21 cttccggaat tcsargtnma gctgsagsag tcwgg 35 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> heavy chain constant region reverse primer <400> 22 ggaagatctg acatttggga aggactgact ctc 33 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> light chain constant region forward primer <400> 23 gggagctcga yattgtgmts acmcarwctm ca 32 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> light chain constant region reverse primer <400> 24 ggtgcatgcg gatacagttg gtgcagcatc 30

Claims (20)

delete delete delete A heavy chain CDR1 according to SEQ ID NO: 3; A heavy chain CDR2 as set forth in SEQ ID NO: 4; A heavy chain variable region comprising heavy chain CDR3 as set forth in SEQ ID NO: 5 and light chain CDR1 as set forth in SEQ ID NO: 6; A light chain CDR2 as set forth in SEQ ID NO: 7; And an antigen-binding portion thereof, which specifically binds to ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase / IMP cyclohydrolase) comprising a light chain variable region comprising the light chain CDR3 of SEQ ID NO: snippet.
5. The fragment of claim 4, wherein the autoimmune antibody comprises an autoimmune antibody or antigen-binding portion thereof comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 1 and the light chain amino acid sequence set forth in SEQ ID NO:
5. The fragment of claim 4 comprising an autoimmune antibody or antigen-binding portion thereof, produced by a hybridoma cell line of Accession No. KCTC 11873BP.
A fragment consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 17 to 20 which is an antigen specifically binding to the autoimmune antibody of claim 4.
delete delete delete Wherein the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 17 to 20, which is an agent for measuring the expression level of an autoimmune antibody or a fragment containing an antigen binding portion thereof specifically binding to ATIC.
A hybridoma cell line producing the autoimmune antibody of claim 4.
13. The cell line according to claim 12, wherein the hybridoma cell line is the accession number KCTC 11873BP.
A kit for the diagnosis of liver cancer comprising the composition of claim 11.
15. The kit according to claim 14, wherein said kit is selected from the group consisting of Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, radial immunodiffusion, Oucheroni immunodiffusion, rocket immunoelectrophoresis, A fixed assay method, a FACS or a protein chip method.
A method for providing information for diagnosis of liver cancer, comprising using the composition of claim 11, detecting a fragment comprising an autoimmune antibody or an antigen binding site thereof that specifically binds to ATIC.
17. The method of claim 16,
(a) measuring the level of expression of a fragment comprising an autoimmune antibody or an antigen-binding portion thereof that specifically binds to ATIC from a sample of a subject suspected of having liver cancer; And
(b) comparing the expression level to an expression level of a normal control sample, the fragment comprising an autoimmune antibody or an antigen-binding portion thereof that specifically binds to ATIC.
18. The method according to claim 17, wherein the sample is any one selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid and intercellular fluid separated from the individual.
18. The method of claim 17, wherein the method for measuring the level of expression of the autoimmune antibody or fragment comprising the antigen binding site thereof comprises the steps of Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Wherein the immunoassay method uses one of the following methods: Teronii immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS or protein chip methods.
(a) measuring the level of expression of an autoimmune antibody or fragment thereof comprising an antigen-binding portion thereof that specifically binds to ATIC, using the composition of claim 11;
(b) administering a candidate substance for the treatment of liver cancer; And
(c) confirming that the expression level of an autoimmune antibody specifically binding to ATIC or a fragment containing the antigen-binding site thereof is decreased as compared to the step (a).

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