KR102128251B1 - Biomarker Composition for Diagnosing Colorectal Cancer Specifically Binding to Arginine-methylated Dopamine Receptor D2 - Google Patents

Abstract

The present invention relates to a biomarker composition and a diagnostic kit for diagnosis of colorectal cancer, which can detect colorectal cancer by using the expression level of arginine-methylated dopamine receptor D2 (DRD2) as an indicator; and to a method for providing information for diagnosis of colorectal cancer. By using antibodies specifically recognizing the arginine-methylated DRD protein, which is a biomarker for diagnosis of colorectal cancer, the expression level of arginine-methylated DRD2 in the blood of a patient suspected of having cancer can be measured, thereby enabling analyses to be simply performed on the blood without performing a biopsy on the patient, wherein a diagnosis of colorectal cancer can be made when the expression of arginine-methylated DRD2 is lower than a normal person.

Description

Biomarker Composition for Diagnosing Colorectal Cancer Specifically Binding to Arginine-methylated Dopamine Receptor D2}

The present invention relates to a biomarker composition for diagnosing colorectal cancer and a diagnostic kit for diagnosing colorectal cancer by using the expression level of DRD2 (Dopamine Receptor D2) methylated with arginine as an index, and a method for providing information for colorectal cancer diagnosis.

Almost all proteins in a cell are synthesized through translation of mRNA, and then modification occurs at a specific amino acid residue, which is called post-translational modification (PTM). Post-translational modifications include phosphorylation, acetylation, methylation, ADP ribosylation, adenylation, uridilation, palmitylation, sugar (chain) addition, amidation, etc., and abnormal occurrence or inhibition of post-translational modification of proteins is a cancer It is known to play an important role in development, progression and metastasis. Recently, antibodies with improved sensitivity and specificity for specific post-translational modifications such as phosphorylation, acetylation, methylation, etc. have been developed, and due to advances in mass spectrometry equipment and analysis technology, and accumulation of databases, modifications of specific amino acid residues in cells or tissues It has been easier to characterize at the protein level using immuno-affinity purification (IAP) and mass spectrometry (LC-MS/MS). In addition, it is possible to effectively discover new biomarkers from proteins contained in cells or tissues by utilizing these technologies.

When methylation of arginine among amino acid residues occurs, a methyl group is transferred to ω-nitrogen present in the arginine residue, and a catalytic enzyme called protein arginine methyltransferase (PRMT) is involved in this reaction. . PRMT is classified into type I PRMT (forms asymmetric dimethylarginine (ADMA)) and type II PRMT (forms form dimethylarginine (SDMA)) according to the final reaction product, all of which deliver one methyl group to arginine residues. The mono-type methylation reaction (monomethylarginine; MMA) is primarily catalyzed (see FIG. 1).

Based on the IAP-LC/MS/MS method for HCT116 cells derived from colorectal cancer, human primary T cells, and human embryonic kidney 293 cells from 2014 to recent, approximately 10,000 sites of single methylation occurred. Was identified. In 2016, sera isolated from patients with acute myeloid leukemia, breast cancer, and non-small cell lung cancer (4 in each cancer type, 12 in total) using specific antibodies to a single methylation reaction, such as IgG and albumin Specific MMA sites of each cancer were identified by performing IAP-LC/MS/MS without removing major serum proteins. These results suggest that the MMA site can be developed as a new biomarker. However, until now, the results of studies that suggested the potential as a biomarker for a single arginine methylation reaction in serum proteins of colorectal cancer patients through systematic experiments and verification have not been reported.

Accordingly, the present inventors have completed the present invention by confirming a change in the expression level of the DRD2 protein in which a single arginine methylation reaction appears as a result of a study on the single-arginine methylation of a protein in serum of normal people and colon cancer patients.

An object of the present invention is to provide a biomarker composition for diagnosing colorectal cancer that specifically binds to DRD2 (Dopamine Receptor D2) in which arginine is methylated.

DRD2 in which the arginine is methylated may be that the 61st arginine residue in the amino acid sequence of SEQ ID NO: 1 is methylated.

In addition, an object of the present invention is to provide a kit for diagnosing colorectal cancer comprising the composition.

The composition may be an antibody that specifically binds to DRD2 in which the 61st arginine residue in the amino acid sequence of SEQ ID NO: 1 is methylated. More specifically, the antibody may be an antibody that specifically binds to an arginine methylation site of DRD2 in which arginine is methylated.

In addition, the present invention comprises the steps of: a) measuring the expression level of DRD2 in which arginine is methylated in a biological sample isolated from a subject; And b) comparing the expression level of the measured arginine methylated DRD2 with a normal control.

The biological sample in step a) may be blood, plasma or serum.

In step a), the expression level of DRD2 in which arginine is methylated may be measured by an antigen-antibody reaction method.

The step b) may be diagnosed as colon cancer when the measured expression level of DRD2 in which the arginine is methylated is lower than that in the normal control group.

<Biomarker composition for colon cancer diagnosis>

According to one embodiment of the present invention, the present invention provides a biomarker composition for diagnosing colorectal cancer that specifically binds to DRD2 in which arginine is methylated.

The DRD2 (Dopamine Receptor D2) has the amino acid sequence of SEQ ID NO: 1 and is a central neuronal receptor that recognizes the neurotransmitter dopamine, and is a peptide that delivers a binding signal with dopamine into cells. These DRD2 proteins are mainly present in the progenitor or pituitary gland as synaptic receptors, and are known to inhibit the release of neurotransmitters or prolactin by inhibiting adenylate cyclase, K ⁺ channel initiation, or Ca ²⁺ channel inhibition. Recently, studies have been conducted to analyze changes in the expression of DRD2 in colorectal cancer patients, but studies on the correlation between methylation of DRD2 and colorectal cancer are still insufficient.

As a result, the present inventors compared the expression level of the protein with methylation of arginine in the serum of normal and colorectal cancer patients, and confirmed that the expression of DRD2 with methylation of arginine in colorectal cancer was lower than that of normal (see Example). . At this time, the arginine methylated DRD2 may have a structure in which the 61st arginine residue in the amino acid sequence of SEQ ID NO: 1 is methylated. That is, by confirming that the structurally modified DRD2 protein acts as an important factor in the development or progression of colon cancer, arginine-methylated DRD2 can be usefully used as a biomarker for diagnosing colon cancer.

Here, the "biomarker" of the present invention is a molecule or compound generally detectable in a biological sample, and refers to an indicator capable of detecting changes in a living body.

The "biomarker composition" of the present invention is a substance capable of specifically binding to a biomarker and measuring its expression level, and may be an antibody, aptamer, peptide nucleic acid (PNA), or the like.

<Kitchen cancer diagnostic kit>

According to another embodiment of the present invention, the present invention provides a kit for diagnosing colorectal cancer comprising a biomarker composition for diagnosing colorectal cancer that specifically binds to DRD2 in which arginine is methylated.

The composition is capable of measuring the expression level of DRD2 in which arginine is methylated by specifically binding to DRD2 having the amino acid sequence of SEQ ID NO: 1, and may be an antibody, aptamer, PNA, or the like. In this case, the antibody that specifically binds to DRD2 is preferably an antibody that recognizes the entire or fragment of DRD2 or the arginine metallization site of DRD2 regardless of the methylation of the protein. The antibody can be recognized without restriction as long as methylation occurs among the arginine residues included in the amino acid sequence of SEQ ID NO: 1, and specifically, the methylation site of the 61st arginine residue.

Here, “specifically binding” means that the binding force to the target substance is superior to that of other substances to the extent that it can detect the presence of the target substance by binding.

“Antibody” of the present invention means a peptide or polypeptide substantially encoded by or derived from an immunoglobulin gene capable of specifically binding to an antigen or epitope, or a fragment thereof. Such antibodies are in complete form with two full-length light chains and two full-length heavy chains, or include functional fragments of antibody molecules. The functional fragment of the antibody molecule means a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, and the like. In this case, the antibody may be a monoclonal antibody or a polyclonal antibody. Such antibodies can be produced by conventional methods known in the art, for example, by fusion methods, recombinant DNA methods, phage antibody library methods, and the like.

The "diagnostic kit" of the present invention means a substance that can be diagnosed by distinguishing a biological sample collected from a suspected cancer patient from a biological sample collected from a normal person. The kit for diagnosing colorectal cancer is for measuring the presence of DRD2 in which the arginine is methylated in the sample, and the amount of the modified protein can be confirmed using an antibody that specifically binds to DRD2. The kit may be applied to a protein identification method based on immunoassay, for example, Western blot, immunoprecipitation (IP) using an antibody that specifically binds to DRD2 in which arginine is methylated. Methods, such as radioimmunoassay (RIA), enzyme-linked immunoabsorbent assays (ELISA), immunohistochemistry (IMH), flow cytometry analysis (FACS), etc. may be applied. Such kits may include materials suitable for performing various immunochemical reactions using antibodies, and may include, for example, lyophilized antibodies, buffers, stabilizers, and inactive proteins. At this time, the antibody may be labeled with radionuclides, fluorescors, enzymes, or the like.

When using this kit to diagnose colorectal cancer, samples collected from suspected cancer patients can be contacted with antibodies that specifically bind to DRD2 in which the arginine contained in the kit is methylated. At this time, the sample may be diluted to an appropriate degree before contact with the antibody, and the antibody may be immobilized on a solid phase to facilitate subsequent steps such as washing or separation of the complex. The solid shape is glass or plastic, and may be, for example, a microtiter plate, rod, bead, microbead, or the like. Further, the antibody may be bound to a probe substrate or protein chip. As described above, a sample and an antibody may be prepared, and the sample may be placed in constant temperature with the antibody, followed by washing to measure the antibody-marker complex formed. This is done by equilibrating the washed mixture with a secondary reagent, preferably a secondary antibody. Measurement of the amount or presence detection of the antibody-marker complex can be achieved through fluorescence, luminescence, chemiluminescence, absorbance, reflection or transmission. In addition to the above method, a marker in a sample can be detected by conducting an indirect analysis method, for example, a competitive or inhibitory reaction analysis with a monoclonal antibody binding to another epitope of the marker.

<Method of providing information for colorectal cancer diagnosis>

According to another embodiment of the present invention, the present invention provides a method for providing information for the diagnosis of colorectal cancer. Specifically, the method comprises the steps of: a) measuring the expression level of DRD2 in which arginine is methylated in a biological sample isolated from a subject; And b) comparing the measured expression level of DRD2 methylated with arginine to a normal control.

The step a) is a process of confirming the presence and expression level of DRD2 in which biomarker arginine is methylated in a biological sample of a suspected cancer patient (subject) to diagnose colorectal cancer. The biological sample may be selected from the group consisting of blood, plasma, serum, colon cells and colon tissue, and is preferably blood, plasma or serum that is easily obtained from a subject. At this time, the amount of protein can be measured by an antigen-antibody reaction method using an antibody that specifically binds to DRD2 in which arginine is methylated. The antigen-antibody reaction is Western blot, immunoprecipitation (IP), radioactive immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunostaining (IHC), flow cytometry (FACS), enzymatic substrate coloration, antigen -It may be performed using an antibody aggregation method, a protein chip, or the like. For example, the expression level of DRD2 in which arginine is methylated can be measured using the kit for diagnosing colorectal cancer.

Step b) is a process of comparing the expression level of DRD2 with methylated arginine measured in a suspected cancer patient to a normal person. At this time, when the measured expression level of DRD2 methylated arginine is lower than that of the normal control group, it can be diagnosed as colon cancer.

“Diagnosis” of the present invention means identifying the presence or characteristics of a pathological condition, determining the susceptibility of an individual to a particular disease or condition, whether or not the individual currently has a particular disease or condition To determine the prognosis of an individual with a particular disease or condition, therametrics (e.g., monitoring the condition of an object to provide information about treatment efficacy) Includes.

In the present invention, by using an antibody specifically recognizing a DRD2 protein in which arginine methylated as a biomarker for colorectal cancer diagnosis is measured, the level of expression of DRD2 in which arginine is methylated in the suspected cancer patient is measured to measure blood levels without biopsy from the patient. As a result, it can be easily analyzed, and a case in which the expression of DRD2 in which arginine is methylated is lower than that in a normal person can be diagnosed as colon cancer.

1 is a schematic diagram of a reaction product of an arginine methylation reaction of a protein and a protein arginine methyl transferase (PRMT) produced therefrom.
2 is a graph measuring the expression level of the normal protein DRD2 in serum of domestic healthy human (HD) and colorectal cancer patients (CRCP) according to one embodiment of the present invention by ELISA.
Figure 3 is a graph measuring the expression level of the normal protein DRD2 in serum of healthy humans (HD) and colorectal cancer patients (CRCP) who have been approved by the US FDA according to one embodiment of the present invention by ELISA.
Figure 4 is a group of each group by measuring the quantitative level of the protein DRD2 arginine methylated in serum of domestic healthy human (HD) and colorectal cancer patients (CRCP) according to one embodiment of the present invention by IAP and LC/MS/MS analysis It is a graph showing normalized intensity per star.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions have been presented by way of example only to help understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.

Example 1. ELISA measurement for normal protein DRD2

Serum obtained from 20 healthy donor (HD) and colorectal cancer patients (CRCP) in Korea, respectively, was measured by ELISA for the level of expression of normal protein DRD2 without methylation of arginine. The results are shown in FIG. 2.

Referring to FIG. 2, it was confirmed that CRCP has a reduced expression level of DRD2 protein compared to HD.

In order to verify the reliability of the above results, the expression level of the normal protein DRD2 was measured by ELISA in the same manner as described above for serum obtained from 20 healthy human (HD) and colorectal cancer patients (CRCP), respectively, who were approved by the US FDA. Did. The results are shown in FIG. 3.

Referring to FIG. 3, it was confirmed that the expression level of DRD2 protein is reduced compared to HD, and CRCP exhibits a similar tendency to that of FIG. 2.

Example 2. Mass spectrometry for protein DRD2 with arginine methylated

Serum obtained from 18 healthy humans (HD) and colorectal cancer patients (CRCP), respectively, was measured by mass spectrometry for the expression level of the protein DRD2 with methylated arginine.

First, each serum obtained from 18 patients with colorectal cancer was classified into three groups of 6 people so that sex, age, and colorectal cancer stages (stages 2 to 4) were uniformly included.

Each serum obtained from 18 healthy humans, the control group, was also divided into three groups of six, so that sex and age matched the group of colon cancer patients as much as possible.

Pooling 6 serums in each group and commissioning a total of three pairs of HD-CRCP serums from Cell Signaling Technology (CST) located in the United States to express arginine methylated protein DRD2 through IAP and LC/MS/MS analysis Levels were measured. The results are shown in Fig. 4 and Table 1 below.

Fold change (healthy person: colorectal cancer patient) Average p value Group 1 (G1) Group 2 (G2) Group 3 (G3) DRD2 -2.8 -2.9 -2.3 -2.7 0.01

Figure 4 shows the normalized intensity of the quantitative level of the DRD2 protein methylated arginine.

Table 1 is a result of dividing the intensity value of CRCP measured in FIG. 4 by the intensity value of HD.

As a result, it was confirmed that the expression level of the DRD2 protein in which arginine is methylated is reduced compared to HD in CRCP.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION Research And Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Biomarker Composition for Diagnosing Colorectal Cancer Specifically Binding to Arginine-methylated Dopamine Receptor D2 <130> PN190031 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 443 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of DRD2 <400> 1 Met Asp Pro Leu Asn Leu Ser Trp Tyr Asp Asp Asp Leu Glu Arg Gln 1 5 10 15 Asn Trp Ser Arg Pro Phe Asn Gly Ser Asp Gly Lys Ala Asp Arg Pro 20 25 30 His Tyr Asn Tyr Tyr Ala Thr Leu Leu Thr Leu Leu Ile Ala Val Ile 35 40 45 Val Phe Gly Asn Val Leu Val Cys Met Ala Val Ser Arg Glu Lys Ala 50 55 60 Leu Gln Thr Thr Thr Asn Tyr Leu Ile Val Ser Leu Ala Val Ala Asp 65 70 75 80 Leu Leu Val Ala Thr Leu Val Met Pro Trp Val Val Tyr Leu Glu Val 85 90 95 Val Gly Glu Trp Lys Phe Ser Arg Ile His Cys Asp Ile Phe Val Thr 100 105 110 Leu Asp Val Met Met Cys Thr Ala Ser Ile Leu Asn Leu Cys Ala Ile 115 120 125 Ser Ile Asp Arg Tyr Thr Ala Val Ala Met Pro Met Leu Tyr Asn Thr 130 135 140 Arg Tyr Ser Ser Lys Arg Arg Val Thr Val Met Ile Ser Ile Val Trp 145 150 155 160 Val Leu Ser Phe Thr Ile Ser Cys Pro Leu Leu Phe Gly Leu Asn Asn 165 170 175 Ala Asp Gln Asn Glu Cys Ile Ile Ala Asn Pro Ala Phe Val Val Tyr 180 185 190 Ser Ser Ile Val Ser Phe Tyr Val Pro Phe Ile Val Thr Leu Leu Val 195 200 205 Tyr Ile Lys Ile Tyr Ile Val Leu Arg Arg Arg Arg Lys Arg Val Asn 210 215 220 Thr Lys Arg Ser Ser Arg Ala Phe Arg Ala His Leu Arg Ala Pro Leu 225 230 235 240 Lys Gly Asn Cys Thr His Pro Glu Asp Met Lys Leu Cys Thr Val Ile 245 250 255 Met Lys Ser Asn Gly Ser Phe Pro Val Asn Arg Arg Arg Val Glu Ala 260 265 270 Ala Arg Arg Ala Gln Glu Leu Glu Met Glu Met Leu Ser Ser Thr Ser 275 280 285 Pro Pro Glu Arg Thr Arg Tyr Ser Pro Ile Pro Pro Ser His His Gln 290 295 300 Leu Thr Leu Pro Asp Pro Ser His His Gly Leu His Ser Thr Pro Asp 305 310 315 320 Ser Pro Ala Lys Pro Glu Lys Asn Gly His Ala Lys Asp His Pro Lys 325 330 335 Ile Ala Lys Ile Phe Glu Ile Gln Thr Met Pro Asn Gly Lys Thr Arg 340 345 350 Thr Ser Leu Lys Thr Met Ser Arg Arg Lys Leu Ser Gln Gln Lys Glu 355 360 365 Lys Lys Ala Thr Gln Met Leu Ala Ile Val Leu Gly Val Phe Ile Ile 370 375 380 Cys Trp Leu Pro Phe Phe Ile Thr His Ile Leu Asn Ile His Cys Asp 385 390 395 400 Cys Asn Ile Pro Pro Val Leu Tyr Ser Ala Phe Thr Trp Leu Gly Tyr 405 410 415 Val Asn Ser Ala Val Asn Pro Ile Ile Tyr Thr Thr Phe Asn Ile Glu 420 425 430 Phe Arg Lys Ala Phe Leu Lys Ile Leu His Cys 435 440

Claims (9)

A biomarker composition for diagnosing colorectal cancer that specifically binds to Arginine methylated DRD2 (Dopamine Receptor D2).
According to claim 1,
The arginine methylated DRD2 is a biomarker composition for diagnosing colon cancer in which the 61st arginine residue in the amino acid sequence of SEQ ID NO: 1 is methylated.
A kit for diagnosing colorectal cancer comprising the composition of claim 1.
According to claim 3,
The composition is a kit for diagnosing colorectal cancer, wherein the composition is an antibody that specifically binds to DRD2 in which the 61st arginine residue in the amino acid sequence of SEQ ID NO: 1 is methylated.
According to claim 4,
The antibody is a kit for diagnosing colon cancer, which is an antibody that specifically binds to the arginine methylation site of DRD2 in which arginine is methylated.
a) measuring the expression level of DRD2 in which arginine is methylated in a biological sample isolated from a subject; And
b) comparing the measured expression level of arginine methylated DRD2 with a normal control
Method for providing information for the diagnosis of colorectal cancer, including.
The method of claim 6,
Method of providing information for the diagnosis of colorectal cancer, wherein the biological sample in step a) is blood, plasma or serum.
The method of claim 6,
The step a) is arginine methylation method of providing information for the diagnosis of colorectal cancer, which is to measure the expression level of methylated DRD2 by an antigen-antibody reaction method.
The method of claim 6,
The step b) is a method of providing information for diagnosing colorectal cancer when the measured level of expression of DRD2 methylated arginine is lower than that of a normal control group.

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