KR101777254B1 - Specific monoclonal antibody from a specific antigen EN2 protein or composition comprising the same for diagnosis of prostate cancer - Google Patents

Abstract

The present invention relates to an EN2 monoclonal antibody specifically identifying EN2 protein. The EN2 monoclonal antibody can confirm existence and nonexistence of EN2 protein in a body and can quantify EN2 protein, and has excellent detection sensitivity compared to existing EN2 protein antibody, thereby being used as a diagnostic composition for prostate cancer. When diagnosing prostate cancer using the antibody of the present invention, the antigen has higher diagnostic validity than a prostate-specific antigen (PSA) detection result in blood which is an existing diagnostic method for prostate cancer.

Description

The present invention relates to a monoclonal antibody obtained from a specific antigen that specifically recognizes an EN2 protein or a composition for diagnosing prostate cancer containing the same,

The present invention relates to a monoclonal antibody obtained from a specific antigen which specifically recognizes an EN2 protein or a composition for diagnosing prostate cancer containing the antibody.

An antibody is an antibody that generally has an epitope that allows an external substance (a substance with a certain size and condition) (antigen) to be injected into an organism and an organism capable of specifically recognizing an external substance through a humoral immune response Antibodies are formed and they are taken from the blood and separated from the serum. The antibody thus formed is a polyclonal antibody having several antigen recognition sites.

In the humoral immune response, the antigen is present in the B cell as a small epitope by various antigen presenting cells (APC) and activates the B cell. In this process, only the genes that make antibodies capable of reacting with the antigen are rearranged, and the remaining unnecessary genes are removed, so that only one antibody is produced in a large amount, and the plasma cells ). Since the antigen generally has several epitopes, different kinds of plasma cells are different, and thus various antibodies are produced. A total of several kinds of antibodies secreted from various kinds of plasma cells (that is, genes that make antibodies) are referred to as 'polyclonal antibodies', and only one type of antibody produced in one kind of plasma cells Clone antibody ".

The diagnostic market using antibodies has been rapidly increasing since 1980 and it has been used for the development and diagnosis of highly efficient diagnostic kits because it can detect very small amount of protein specifically expressed by disease or symptom . For this purpose, it is essential to develop antibodies with both specificity and sensitivity to proteins (antigens) expressed by diseases and symptoms. In order to generate antibodies with specificity and sensitivity, the efficiency of the antibody can be maximized by some consideration. First, by comparing the primary sequence of the antigen to be generated, it is possible to maximize the immune response by selecting a highly heterogeneous animal species and immune animal. For example, when a sequence of a human protein is used, an antibody having high potency can be obtained by selecting an animal having high sequence and heterogeneity to induce an immune response. Secondly, considering the nature of the antigen and the quality of the antigen as a function of the steric structure in the sequence after modification after translation, it is considered to be selected. Particularly when the whole protein is not used as an antigen but when it is used as an antigen in the form of some peptides, it should be selected especially. Third, the antibody should be prepared according to the purpose. In the case of polyclonal antibody, it is easy to produce, it is easy to detect the antigen due to various epitopes, and it has a wide selection of immune animals according to the antigen. However, antibody specificity drops and it is not easy to mass-produce the same activity. Monoclonal antibodies have the advantage of producing antibodies in culture supernatant, which can produce large quantities of antibodies having the same specificity of activity. However, it takes a long time to produce antibodies, limited immune animals, It is not suitable. Therefore, monoclonal antibodies can be produced when mass production of an antibody having the same activity is required after confirming specificity of an antigen through production of polyclonal antibodies.

A prostate is a biliary-sized male reproductive organs beneath the bladder, in front of the rectum, that produces and stores part of the semen. The upper part is fixed to the bladder neck, ie, the part of the bladder to the urethra, adjacent to the anterior tibial prostate ligament, and the lower part is fixed by the genitourinary diaphragm. Most of the cancer that occurs in the prostate gland is adenocarcinoma (cancer of the gland cell) that occurs in the prostate gland. The types can be classified according to the degree of differentiation of the tumor tissue and the characteristics of the cells. Prostate cancer is one of the most common urological tumors worldwide. In the United States, about 180,890 new prostate cancers were diagnosed in 2016, accounting for 10.7% of all US new diagnoses, followed by breast cancer and lung cancer for the third time It is a frequently occurring tumor. According to statistics from 2009 to 2013, 129.4 out of 100,000 men worldwide have prostate cancer and the mortality rate for prostate cancer is 26,120 in 2016 statistics. Prostate cancer is also uncommon before age 50, but it is increasing rapidly after age 50. Recently, the prolongation of the life expectancy in Korea has necessitated continuous management in order to prevent the aging of the elderly and prognosis of prostate cancer. In particular, human prostate cancer appears to have a tendency to metastasize to bone and is known to increase the mortality rate of patients by inevitably progressing from an androgen-dependent state to an androgen-resistant state. Furthermore, about 25% of men who have received prostate cancer treatment are those that require additional treatment due to recurrence of disease, which is currently the second leading cause of cancer deaths in the United States in the United States. .

One of the currently used diagnostic methods for prostate cancer is direct imaging of the prostate gland or biopsy. In the case of direct diagnosis or diagnosis through biopsy, it is difficult to diagnose the onset of prostate cancer at an early stage, and it is urgent to develop a method that can diagnose it in vitro. Indirect methods include the use of a prostate-specific antigen (PSA) test to diagnose in vitro. However, the PSA used for diagnosis has been produced not only in the malignant prostate epithelium but also in the normal and positive tissues, resulting in an increase in the false positive rate in the detection of prostate cancer. In addition, a very high serum PSA level can be used as a standard diagnostic tool for effective prostate cancer, while a weak PSA serum level of 2-10 ng / ml can lead to a definite diagnosis of prostate cancer I can not. The PSA assay for diagnosing prostate cancer is problematic in terms of detection specificity because the serum PSA in such a weakly rising state may be derived from a non-tumor disease such as benign prostatic hyperplasia (BPH), prostatitis or other physical trauma . Therefore, the diagnosis of prostate cancer using a new biomarker has become a major subject and research has been conducted (Korean Patent Laid-Open No. 10-2009-0111307). Recently, EN2 (Engrailed-2) has been used as a biomarker to diagnose prostate cancer. EN2 is a biomarker capable of solving the problem of detection specificity of PSA (prostate specific antigen) (bio-marker). The EN2 protein acts as a transcription factor in the cell and is overexpressed only in the prostate cancer cell, resulting in a DNA transcriptional regulatory disorder. Furthermore, when EN2 expression increases in prostate cancer cells, the amount of EN2 protein excreted in the urine is also increased, which is suitable for in vitro analysis. Thus, US Pat. Nos. 8460882, 2012-532621 or USP 8722643 disclose techniques relating to a diagnostic composition for recognizing EN2 protein, and Korean Patent Laid-Open No. 10-2016-0077788 Techniques relating to DNA plasmids that specifically bind EN2 are disclosed.

Accordingly, the inventors of the present invention studied a composition relating to prostate cancer diagnosis, and produced a monoclonal antibody which can more effectively diagnose EN2 protein. The present invention can be completed by manufacturing a prostate cancer diagnostic composition using the antibody.

U.S. Patent No. 8460882 entitled Cancer biomarkers, Applied to: The University of Surrey, US Patent No. 8722643 entitled Targeting EN2, PAX2, and / or DEFB1 for treatment of prostate conditions, filed by Phigenix, Inc., U.S. Patent No. 7358045 (entitled "Methods for Determining Development of Breast Adenocarcinoma or Breast Inflammatory Carcinoma," Applied to INST RECH S CLINIQUES DE MONTR, Apr. 15, 2008) Japanese Patent Laid-Open Publication No. 2012-532621 (entitled Therapeutic Peptides, Polypeptides and Nucleic Acid Arrays, Applicant: The University of Surrey, Dec. 20, 2012) Korean Patent Laid-Open No. 10-2009-0111307 (entitled DNA Platamer specifically binding to EN2 and its use, Applicant: Industry & Academy Collaboration Foundation, POSTECH, Publication Date: 2016.07.04) Korean Patent Laid-Open No. 10-2016-0077788 (the title of the invention: use of the prostate-specific transcript and its use in the diagnosis and treatment of prostate cancer, applicant: Exxon Heat Practicals, Inc., Disclosure Date: Oct. 26, )

It is an object of the present invention to provide a monoclonal antibody obtained from a specific antigen which specifically recognizes EN2 protein or a composition for diagnosing prostate cancer containing the antibody.

The present invention provides a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or 3; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 5, 6 or 7.

The antibody is also characterized by recognizing an epitope consisting of the amino acid sequence of SEQ ID NO: 10.

The monoclonal antibody of the present invention is characterized by recognizing EN2 (Engrailed-2, Accession No. NP - 001018.2) protein or its recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1.

Wherein the antibody is an immunoglobulin G type.

The present invention can also provide a diagnostic composition for diagnosing the presence or absence of EN2 (Engrailed-2) protein comprising said monoclonal antibody or a prostate cancer diagnostic agent.

The present invention also relates to a hybridoma cell line that is a Accession No. KCLRF-BP-00405 and provides a monoclonal antibody produced by said hybridoma cell line.

Hereinafter, the present invention will be described in detail.

The EN2 protein, the recombinant protein thereof, the fragment of the EN2 protein and the amino acid sequence information contained in the antibody recognizing the EN2 protein used in the present invention are as follows.

The protein consisting of the amino acid sequence of SEQ ID NO: 1 below relates to the EN2 protein expressed in the mammal, especially the human body.

SEQ ID NO: 1 : NCBI Reference Sequence Accession No. NP_001418.2

The amino acid sequences of SEQ ID NOS: 2 to 7 below are amino acid sequences constituting the monoclonal antibodies of the present invention. The antibody comprising the variable heavy chain or variable light chain sequence of SEQ ID NOS: 2 to 7 below is a monoclonal cell obtained by fusion of an immunocompetent lymphocyte with an EN2 peptide consisting of the amino acid sequence of SEQ ID NO: 9 and myeloma cells (Accession number KCLRF-BP-00405). The underlined part of each sequence is the sequence of CDR3.

SEQ ID NO: 2 : Variable heavy chain amino acid sequence of EN2 monoclonal antibody

SEQ ID NO: 3 : Variable heavy chain amino acid sequence of EN2 monoclonal antibody

SEQ ID NO: 4 : Variable light chain amino acid sequence of EN2 monoclonal antibody

SEQ ID NO: 5 : Variable light chain amino acid sequence of EN2 monoclonal antibody

SEQ ID NO: 6 : Variable light chain amino acid sequence of EN2 monoclonal antibody (antigen: EN2 peptide consisting of amino acid sequence of SEQ ID NO: 15)

SEQ ID NO: 7 : Variable light chain amino acid sequence of EN2 monoclonal antibody (antigen: EN2 peptide consisting of amino acid sequence of SEQ ID NO: 15)

(In the amino acid sequence of SEQ ID NO: 7, X may be substituted by all amino acids)

The amino acid sequence of SEQ ID NO: 8 below is the amino acid sequence constituting the EN2 recombinant protein used for the identification of the monoclonal antibody in the present invention. The EN2 recombinant protein comprising the amino acid sequence of SEQ ID NO: 8 below contains the T7 tag (blue) to enhance the expression efficiency of the EN2 protein (Accession No. NP_001418.2) and contains His tag (yellow) to facilitate purification . Also, for mass culture using E. coli, E. coli expression vector was used for gene recombination. For this purpose, a restriction enzyme site (light blue) is used.

SEQ ID NO: 8 : Recombinant EN2 protein

SEQ ID NO: 9: fragment (peptide) of EN2 protein for producing the antibody of the present invention

CTRYSDRPSSGPRSRKPKKKNPNKEDKRPR

SEQ ID NO: 10: An epitope recognized by the antibody of the present invention

DRPSSGPRSRKPKKK

The monoclonal antibody of the present invention recognizes the EN2 protein. The EN2 protein may be extracorporeally extracted from a mammalian body, or may be a recombinant EN2 protein. Thus, the present invention can confirm or quantify the presence or absence of EN2 protein in the body of a patient with prostate cancer. That is, the present invention can provide a method of diagnosing the presence or absence of EN2 protein by specifically recognizing EN2 protein using the above monoclonal antibody. At this time, the presence or absence of the EN2 protein can be confirmed by any conventional experimental method used for the presence or the quantification of the existing protein.

The present invention also provides an EN2 monoclonal antibody composition which specifically recognizes an EN2 protein or a diagnostic agent containing the EN2 monoclonal antibody composition. The diagnostic agent may include a labeled secondary antibody, a chromophore, an enzyme conjugated with the antibody, and other substances capable of binding to the substrate or antibody.

Meanwhile, the diagnostic agent of the present invention may include a recombinant EN2 protein capable of quantifying the EN2 protein contained in the body of the subject to be diagnosed.

Generally, an antibody refers to a specific protein molecule directed against an antigenic site. Thus, the antibody of the present invention may preferably comprise a monoclonal antibody that specifically binds to the EN2 protein.

The monoclonal antibodies of the present invention can be prepared using the hybridoma method (Kohler G. and Milstein C.) or the phage antibody library (Clackson et al. Marks et al.) Technique, as is well known in the art to which the present invention pertains. . In order to carry out the hybridoma method, cells of an immunologically appropriate host animal such as a mouse and a cancer or myeloma cell line can be used. After these two types of cells are fused with each other by a method using polyethylene glycol or the like, that is, a method widely known in the technical field to which the present invention belongs, the antibody producing cells can be proliferated by a standard tissue culture method . Subsequently, a uniform cell population is obtained by subcloning by a limited dilution technique, and then a hybridoma capable of producing an antibody specific for the recombinant protein of the present invention can be produced in vitro or in vivo Lt; / RTI > The above-described phage antibody library method comprises the steps of obtaining an antibody gene for a recombinant protein of the present invention and expressing it in the form of a fusion protein on the surface of a phage to prepare an antibody library in vitro, A monoclonal antibody that binds to a protein can be isolated and produced. The antibodies produced by the above methods can be separated by methods such as centrifugation, electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.

The present invention provides a method for producing a monoclonal antibody using an immunogenic fragment peptide (EN2 peptide) of EN2 protein. The EN2 peptide is a fragment of EN2 protein having at least one epitope that can be recognized by an antibody to the EN2 protein that increases or decreases in the body of a patient with prostate cancer and is expected to be highly antigen-antibody reactive through a programming method Lt; RTI ID = 0.0 > amino acid < / RTI >

The EN2 monoclonal antibody of the present invention can be prepared by injecting an animal with an immunogenic fragment peptide of EN2 protein as an antigen and then isolating the spleen tissue and using fusion technology with myeloma cells. The animal may be any animal host such as a mouse or a rat.

More specifically, the following method may be used as a method for producing the antibody of the present invention.

Preferably (step 1) preparing an immunogenic fragment peptide of the EN2 protein as an antigen for producing an antibody;

(Step 2) mixing and emulsifying the antigen and the adjuvant;

(Step 3) injecting the animal into which the adjuvant is emulsified with the antigen at intervals of 5 to 20 days 2 to 6 times;

(Step 4) After 7 to 20 days from the administration of the antigen, serum is separated from the animal, the activity of the antibody is confirmed, and then the spleen of the animal is separated.

(Step 5) A lymphocyte is isolated from the spleen, mixed with myeloma cells to induce cell fusion, and monoclonal cells producing an antibody having a high antigen-antibody reaction to EN2 protein or EN2 recombinant protein among fused cells Selecting;

(Step 6) culturing the selected monoclonal cells to obtain immunoglobulin from the culture medium; And

(Step 7) separating and purifying the immunoglobulin;

. ≪ / RTI >

The antibody may comprise a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab) 2, F (ab) 2, Fv and the like.

In the above-mentioned method for producing an antibody, any animal can be used as an animal which can cause an immune response, but it may be preferable to use a mouse most easily used for production of a monoclonal antibody. The immunoglobulin of the sixth step may be any type, but preferably it may be immunoglobulin G.

The present invention also provides a method of diagnosing prostate cancer using an EN2 protein or a recombinant protein thereof or an antibody specifically recognizing the same.

(1) separating the body protein from the sample to be analyzed;

(Step 2) contacting the sperm protein of Step 1 with the antibody of the present invention to form an antigen-antibody complex; And

(Step 3) Detecting and analyzing the antigen-antibody complex produced in Step 2 in a quantitative manner.

In the above diagnostic method, the sample of the first stage may be extracted from a subject to be confirmed whether or not prostate cancer has occurred or progressed, and preferably may be tissue, cells, etc. of a mammal (human or other animal) May be urine, blood, plasma, serum, liver cells, and the like.

The step of separating the body protein in the above step 1 can be carried out using a known process, and the amount of the body protein can be measured by various methods known to those skilled in the art.

The two-step antigen-antibody complex means a specific combination of an EN2 protein (or a recombinant protein of the present invention) contained in a body protein in a sample and an antibody. That is, the antigen in the complex means EN2 protein.

That is, the amount of the antigen-antibody complex formed in the control group and the amount of the antigen-antibody complex formed in the subject in which the presence or absence of the prostate cancer is confirmed can be compared through the above-described analysis method, and the presence or absence of the prostate cancer Of prostate cancer can be directly diagnosed by judging the amount of EN2 protein expression.

The EN2 content of the sample to be inspected for the presence or absence of progression of prostate cancer in step 3 can be confirmed through a standard value of the concentration of the recombinant EN2 protein used in the present invention.

The amount of EN2 protein expressed in the presence or absence of progression of prostate cancer is determined by whether it corresponds to 3.1 to 65.4 nM (Sci.Rep. 2013; 3: 2059), which is known as urine EN2 protein concentration range in patients with prostate cancer .

The formation amount of the antigen-antibody complex can be quantitatively measured through the signal size of the detection label. The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule and a radioactive isotope, but is not limited to the substance described above. When the enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase , Acetylcholinesterase, glucose oxidase, and hexokinase, but are not limited to the range described above. Examples of the fluorescent material include fluorescein, phycocyanin, fluorescamine, and the like, but the material is not limited to those described above. Examples of the ligand include, but are not limited to, biotin derivatives and the like. The luminescent material includes, but is not limited to, luciferin. The fine particles include, but are not limited to, colloid, gold, and the like. Examples of the redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, and hydroquinone. Such radioisotopes include, but are not limited to, ³ H, ¹⁴ C, and the like.

The diagnostic method can be performed by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, Immunocytochemistry, immunoprecipitation, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, and the like. However, the method is not limited to the method described above no.

The subject to be diagnosed using the antibody of the present invention can be any subject whose EN2 protein is produced in the body, and it is possible to diagnose all mammals including human. The mammal may be any kind of mammal such as a person, a dog, a cat, a rabbit, a cattle, a goat, a pig, and a horse.

The present invention relates to an EN2 monoclonal antibody that specifically recognizes an EN2 protein. The EN2 monoclonal antibody can be used as a diagnostic agent composition for prostate cancer because it can detect the presence or absence of EN2 protein present in the body and is significantly superior in detection sensitivity to a conventional EN2 protein antibody. When diagnosing prostate cancer using the antibody of the present invention, it is confirmed that diagnosis efficiency is higher than that of prostate-specific antigen (PSA) detection in blood, which is a conventional method of diagnosing prostate cancer. As related prior art, US Patent No. 8460882 also discloses information about EN2 antibody, but EN2 monoclonal antibody having the amino acid sequence as disclosed in the present invention or EN2 monoclonal antibody prepared from the hybridoma cells of the present invention Can be said to be a novel composition completely different in technical characteristics from those disclosed in the prior art.

Fig. 1 (A) is a schematic representation of the cloning of an EN2 (homeobox protein engrailed-2) protein in a large amount by using an E. coli expression vector in Example 1-1, and B is a large- And purified and purified recombinant EN2 protein by SDS-PAGE.
FIG. 2 (A) is the HPLC result obtained by synthesizing and purifying peptides expected to have high antigenicity of EN2 (homeobox protein engrailed-2) in Example 1-2, and B shows the result of mass spectrum of this EN2 peptide Results.
FIG. 3 shows the results of immunization of the polyclonal antibody (FIG. 3A) obtained in Example 2-2 with the EN2 peptide prepared in Example 1-2 as an antigen and the fusion of lymphocytes and myeloma cells of the immunological animal producing the polyclonal antibody (FIG. 3B) obtained from the hybridoma cells obtained in Example 3-3 obtained through the above-described method. In the following figures, the polyclonal antibody is represented by Anti-EN2 (Poly), and the monoclonal antibody of the present invention is represented by Anti-EN2 (Mono).
Figure 4A shows the selective detection of prostate cancer cell line PC3 and LNCaP expressing EN2 protein for the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and the comparative group polyclonal antibody (Anti-EN2 blot experiment, and B is a numerical result.
FIG. 5A shows the quantifiable concentration of the antigen-detectable recombinant EN2 protein against the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and the comparative group polyclonal antibody (Anti-EN2 , And B is the result of quantification.
Figures 6A and 6B show the LNCap prostate cancer cell lines, C and D, against the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and the comparative group Polyclonal Antibody Anti-EN2 (Poly), in the PC3 prostate cancer cell line (EN2) protein by using a Confocal Laser Scanning Microscope (CLSM).
FIG. 7A shows the result of ELISA (EN2 protein quantification standard line) for the selective detection of EN2 protein of the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention and B using the result of A ELISA shows the results of quantitation of EN2 protein in a sample (urine) of actual benign prostatic hyperplasia (P1), prostatitis patient (P2) or prostate cancer patient (P3 ~ P7).
FIG. 8A shows the use of the EN2 monoclonal antibody (Anti-EN2 (Mono)) of the present invention to determine whether the sample (urine) of the actual patients P1 to P7 can distinguish between prostate cancer and prostatitis or benign prostatic hyperplasia western blot method, and B is the prostate-specific antigen (PSA) concentration of these patients.
FIG. 9 shows the results of analyzing the amino acid sequences of the light chain and the heavy chain of the monoclonal antibody prepared in the present invention.
Fig. 10 shows the epitope sequence predicted through epitope mapping using the monoclonal antibody of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

Example 1 Recombinant EN2 protein cloning, expression, purification and preparation of antigen < RTI ID = 0.0 >

Example 1-1. Recombinant EN2 protein cloning, expression, purification

For in-vitro quantification of EN2 protein, recombinant EN2 protein was prepared. In order to maximize protein expression, T7 Tag was used for the N terminal of the protein, and then His Tag was used for N and C terminal to facilitate protein purification. In addition, restriction sites of BamH1 and Xho1 were used for insertion into pET 28a plasmid. Figure 1 (a) illustrates the pET28b / EN2 plasmid. The pET28b / EN2 plasmid was transformed into E. coli (BL21 / DE3) and overexpressed EN2 protein at 0.1 mM IPTG and 37 ℃. Overexpressed E. coli cells were sonicated with lysis buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 × protease inhibitor cocktail, 1 mg / ml lysozyme) and centrifuged to obtain only water-soluble proteins. After incubation with Ni-NTA agarose bead for binding to EN2 / His tag and Ni, the eluate buffer (20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 300 mM imidazole, 1x protease inhibitor cocktail) The imidazole was removed by dialysis (cut off 10K) in a storage buffer (50 mM Tris-HCl pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol) Bicinchoninic acid) and the absorbance at 280 nm.

Examples 1-2. EN2 Peptide Amino Acid Sequence Selection, Peptide Synthesis and Transport Protein Binding

Thirty amino acids of the entire EN2 protein sequence were cut into the same peptide units using the Antigen profiler peptide tool (Thermo Fisher Scientific), each of the peptides was scored with an antigenic index and scored from 1 to 5 And a score of 3.8 or higher was selected. At this time, the antigenic index scored the potential as an antigen due to structural influences and stereostructures according to post-translational modification of nucleotides to proteins.

Among the selected regions, the regions where the hydrophobic residues are distributed are excluded from the selection because the external exposure is blocked in the folding of the proteins. The sequence of the peptides selected according to the above criteria is the highest 10 The peptide with the least hydrophobic residue was selected.

10 mg of the peptide was submitted for synthesis at 98% purity (Anisen, Korea) and a peptide (3570 Da) consisting of the following amino acid sequence was obtained. The result of purifying the peptide by HPLC was shown in FIG. 2A, and the result of mass spectrum was shown in FIG. 2B.

Antigenic peptides for antibody production: CTRYSDRPSSGPRSRKPKKKNPNKEDKRPR

On the other hand, when the peptide is small in size and used as a direct antigen, it is difficult to induce an immune response. Thus, a carrier protein (KLH (keyhole limpet hemocyanin)) was cross-linked using a cross-linker.

For this purpose, peptides, carrier proteins, and cross-linking agents were dissolved at a weight ratio of 1: 1: 1 and sufficiently reacted at room temperature for 2 hours or more. EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) was used as a crosslinking agent and 0.1 M MES (2- [N-morpholino] ethane sulfonic acid (Where the buffer serves to induce the hydrolysis of the amine group).

After the reaction was completed, the separation was carried out on a Thermo Scientific Zeba Spin Desalting Column (# 89891) with a size difference (KLH 400 Kd, 3.5 Kd of peptide).

This EN2 peptide conjugated with the transporter protein was then used as an antigen for preparing the antibody of the present invention.

Examples 1-3. Preparation of adjuvant and emulsified antigen

In order to administer the antigen to the immune animal, it is necessary to bond with the adjuvants so that the antigen having a high solubility in the living body can stay for a long time, thereby preparing a composition in which the adjuvant and the antigen are emulsified. In order to effectively induce immunity, the antigens prepared in Example 1-2 were administered four times in total. To maximize the immune response, the first administration antigen was mixed with Freund's complete adjuvant (FCA) containing killed mycobacterium And Freund's incomplete adjuvant (FIA), which excluded dead mycobacterium, was prepared by mixing the remaining three antigens for administration. In order to effectively emulsify the water-soluble antigen and the hydrophobic adjuvant, the antigen and the adjuvant are mixed using a probe sonication method, and the mixture is kept at 4 ° C so that heat is not applied to the antigen during the mixing. Antigen and adjuvant were mixed in a 1: 1 volume.

Thereafter, in order to prepare a monoclonal antibody having a good reactivity to the EN2 protein, a process for screening a hybridoma cell that produces a monoclonal antibody from the lymphocyte of the animal is selected and an immunized animal that produces a serum with good antibody reaction is selected The experiment of Example 2 was conducted.

&Lt; Example 2: Induction of an immune response using an antigen &

Example 2-1. Selection of immune animals

A mouse (BALB / C) was used to induce an immune response to human EN2 protein sequence. Immunoreactivity was induced in two 5-week-old females against the antigen (peptide mixed with the adjuvant of Example 1-3) in consideration of the individual specificity, since the degree of immune response may vary depending on individuals. The same kind of mice were used as immune animals used for the production of monoclonal antibodies.

Example 2-2. Induce the immune response and check the activity of the antibody

For the first antigen administration, 100 μl of the emulsion was injected into the plantar bones of experimental mice (BALB / C) to induce an immune response. Two weeks after the first injection, 100 μl of the same antigen was administered to the plantar feet of the experimental mice to induce an immune response. Re - injection was performed up to 4 times at intervals of 1 week. After immunization, a small amount of serum was collected and the activity of polyclonal antibody in serum was confirmed by ELISA test.

To this end, ELISA confirmation antigen (EN2 recombinant protein) was diluted to 5 ㎍ / ㎖ in coating buffer, and 50 ㎕ per well was coated on 96 well plate and reacted overnight at 4 ℃. The following day, the cells were blocked with 2% skim milk for 1 hour, diluted to the primary antibody, and reacted at 37 ° C for 2 hours. The reaction plate was washed three times with TBS-T, then diluted with 1: 10,000 ratio of goat anti-mouse IgG conjugated with HRP, and 50 μl of each was diluted in the plate well and reacted at 37 ° C for 1 hour. After completion of the reaction, the plate was washed five times and 50 μl of TMB solution was added to each plate well. After 5 minutes of reaction, 100 μl / well of 1N H ₂ SO _{4 was} added to terminate the reaction. Absorbance was measured at 450 nm using a microplate reader My titer was confirmed.

&Lt; Example 3: Fusion and cloning of lymphoid cells with myeloma cells >

Example 3-1. Fusion - Preparation of hybridoma cells

Lymphocytes were isolated from the immunized animals producing antibodies with high titers based on the titer in the serum of the immunized mice identified in Example 2, and cell fusion was performed.

For this, mouse lymph nodes were removed without damage and washed with DMEM (Dulbecco / Vogt modified Eagle's minimal essential medium) medium. Lymphocytes were separated and dispersed in DMEM medium in a 60 mm dish into single cells. / 0 Ag 14 - ATCC # CRL-1581) and lymphocytes were mixed at a ratio of 1: 1 to the number of cells. Mixed cells were washed 2-3 times with ECF buffer (0.3M mannitol, 0.1mM calcium chloride, 0.1mM magnesium chloride) 10 ~ 20ml, centrifuged to discard the dispersion, and the remaining cell pellet was equilibrated in ECF buffer The cell fusion was induced by applying an electrode of about 1.3 volts in an electrode container. Cells were plated at a density of 200 μl per well in a 96-well plate (2.5 × 10 ⁷ cells / plate) and cultured in a CO ₂ incubator. Fusion After 5 days, 50 μl of HAT media (hypoxanthine-aminopterin-thymidine medium) was added to each well. After 12 days of fusion, colony was identified and the reaction with EN2 recombinant protein (ELISA confirmation antigen) was analyzed by ELISA (Asb. 450 nm) method. The procedure was the same as in Example 2.

Example 3-2 Cloning: Hybridoma selection for producing an antibody having excellent antigen-antibody reactivity

In order to select fusion cells, the positive selection criterion of the ELISA performed in Example 3-1 is an OD value of 1.0 or more (OD value of 1 or less means that the affinity of the actual monoclonal antibody is not good after the next cloning step , And non specific binding also occurs in some OD values, so it is better to select a minimum value of 1 or more.) Since most of the values are more than 0.5 in the confirmation result, more than 0.5 positive And monoclonal cells were selected.

For this, cloning was performed to select monoclonal cells from selected wells based on the ELISA OD value after cell fusion. The cells were transferred to 24 wells and subjected to ELISA test. The cells were spun down and dispersed with HT media. Cell counting was performed so that 150 cells per 96-well plate were dispensed. After 10 to 12 days, ELISA test (the same method as in Example 3-1) was performed, and a second colony was selected by selecting an ELISA test result with a low colony and a high OD value (ELISA positive selection criteria The same as in Example 3-1). Thereafter, further cloning was continued, and the respective cloning procedures were the same as in Example 3-1. From the fourth round of cloning, complete media (DMEM, 10% FBS [Fetal bovine serum] + 1% PS [Penicillin-Streptomycin]) was used.

On the other hand, when the deviation between the positive wells was less than 0.2 (based on the OD value), final cloning was performed for selection of the monoclonal cells. If it was judged that the second cloning matched the selection conditions of the monoclonal cells, And final culture for monoclonal cells was performed. If the cells were grown, they were scaled up with 75T flasks and 150T flasks. After the cells were grown as desired, they were frozen at 3 × 10 ⁶ cells / ml (freezing media composition: 25 ml FBS + 20 ml DMED + 5 ml DMSO ).

&Lt; Example 4: Purification of immunoglobulin G (IgG) >

Example 4-1. Collection of monoclonal cell culture media

The cells obtained in Example 3-3 were dissolved at 37 ° C and cultured in DMEM / 10% FBS / 1% PS medium for 10 days in a 5% CO ₂ humidified incubator. Subsequently, subculture was carried out at intervals of two days to collect the culture medium Respectively.

Example 4-2. Immunoglobulin G (IgG) Isolation

Since the culture medium contains various proteins including FBS, it was decided to purify only immunoglobulin G (IgG) in order to increase the activity and specificity of the antibody.

For this, the culture medium was bound to packed resin (Protein G Sepharose 4 Fast Flow-GE Health care combined with protein G binding specifically to mouse IgG) and washed with a buffer of 2-3 times bead volume Followed by elution. Elution buffer used pH 2-3 buffer to break the specific binding between protein G and IgG. Tris (pH 9) was used to immediately raise to normal pH after elution. Elution was divided into 10 fractions of 1 ml each after 3 ml Elution per resin. Using 10% SDS-PAGE, the fractions were mixed with 9 μl of fraction and 3 μl of 4x sample buffer (containing reducing agent) After boiling for minutes, they were loaded. After electrophoresis, staining was carried out through coomassie brilliant blue r-250 to confirm the fraction. Through this process, the antibody IgG of the present invention was obtained from the fraction denoted as red in FIG. 3. 3, A (Polyclonal Clone) is a polyclonal antibody prepared using a peptide used as an antigen in the present invention, and B is an antibody fragment obtained from a monoclonal cell (hybridoma) producing the antibody of the present invention . The antibody of FIG. 3A was used as a comparative group in the subsequent experiments. The Polyclonal Clone antibody was labeled as Anti-EN2 (Poly), and the monoclonal antibody of the present invention was labeled Anti-EN2 (Mono).

Each antibody (IgG) finally obtained was diluted in PBS / 0.2% sodium azide / 20% glycerol buffer, dispensed, and stored at -80 ° C.

<Example 5> Evaluation of EN2 protein detection ability of EN2 monoclonal antibody in prostate cancer cell line>

EN2 expression is high prostate cancer cell line PC3 (® CRL-1435 ™) and LNCaP (® CRL-1740 ™) the purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) and this 37 ℃, 5% CO ₂ (FBS) and 1% penicillin / streptomycin (P / S) were added to RPMI-1640 (PC3) or RPMI-1640 (LNCaP) medium containing 25 mM HEPES in a humidified incubator.

To confirm the response to EN2 monoclonal antibody, the PC3 and LNCaP cells were cultured for 2 days in a 100 cm ² culture dish for 2 days at a density of 2 × 10 ⁶ cells. Cells were collected by washing twice with cold PBS (phosphate buffered saline) , protease inhibitor cocktail was added to dissolve the cells in ice for 30 minutes. The cell lysate thus obtained was centrifuged at 13,000 rpm for 20 minutes. The supernatant was collected and the concentration of the protein was measured by BCA (bicinchoninic acid assay). The lysed proteins were suspended in 40 μg / well and 20 μg / well 20 μl of each well was added to each well and separated by 4-15% SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). PAGE were transferred to a PVDF membrane and each EN2 antibody (6.6 nM) (Anti-EN2 (Poly), Anti-EN2 (Mono)) was added to 5% skim milk / TBS-T (Tris-saline + Tween20) The HRP-conjugated anti-mouse antibody was diluted with 5% skim milk / TBS (1: 2000) at 4 ° C. overnight, washed three times with TBS-T the following day, -T at a ratio of 1: 2000 and reacted at room temperature for 2 hours. The membranes washed three times with TBS-T were sensitized to X-ray film by using ECL (enhanced chemiluminescence) solution, in which luminol, a substrate of peroxidase bound to the secondary antibody, was oxidized by peroxidase and turned blue . The results are shown in FIG. 4, where FIG. 4A is a band image obtained by performing Western blotting, and FIG. 4B is a graph showing the result of the Western blotting.

4, anti-EN2 (mono) of the present invention is superior to EN2 protein in all two prostate cell lines. However, anti-EN2 (poly) It is confirmed that the antigen-antibody reaction does not appear. This may be a disadvantage in the process of producing polyclonal antibodies because the non-reactive antibodies may be produced according to the degree of antigen-antibody reaction in the blood depending on the immune animal. In addition, the use of polyclonal antibodies may not be useful for quantitative analysis of EN2 proteins, since antigen-antibody activity may vary for each immune animal. On the other hand, when a monoclonal antibody is used as in the present invention, it is very effective in that a large quantity of antibodies of the same quality can be produced through hybridoma cells.

Example 6. Evaluation of antibody sensitivity to recombinant EN2 protein - Western blot < RTI ID = 0.0 >

(1 ng / μl) (24 nM) up to a concentration of at least 1 ng / μl from a maximum of 20 ng / 20 μl (24 nM) using the recombinant EN2 protein used in Example 1-1 as a detection protein instead of the protein extracted from a prostate cancer cell line. (0.05 ng / mu l) (1.2 nM), and the sensitivity of the antibody was confirmed under the same conditions as in Experimental Example 1 above. The results are shown in FIG. 5, where FIG. 5A is a band photograph obtained by performing Western blotting, and FIG. 5B is a graph showing the result of the Western blotting.

Referring to FIG. 5, it is shown that EN2 protein is detected at a concentration of at least 1 ng / 20 μl (0.05 ng / μl) (1.2 nM) in Anti-EN2 (mono) , The antigen-antibody reactivity is excellent and the protein-detecting ability of the monoclonal antibody is excellent. This indicates that EN2 protein can be detected sensitively even when applied to actual urine samples (urine EN2 protein concentration range of prostate cancer patients: 3.1 to 65.4 nM: Sci.Rep. 2013; 3: 2059).

&Lt; Example 7: Evaluation of antibody sensitivity to prostate cancer cell line - Immunofluorescent staining method >

The intracellular EN2 protein of prostate cancer cell lines LNCap and PC3 was detected by immunofluorescence staining. Anti-EN2 (Mono) and anti-EN2 (Poly) were used as the primary antibodies and anti-EN2 (Poly) as the secondary antibodies. Anti-mouse IgG / 594 was used as the secondary antibodies. Binding capacity was analyzed by Confocal Laser Scanning Microscope. The fluorescence staining photographs thereof are shown in Fig.

Referring to FIG. 6, it was found that Anti-EN2 (mono) was significantly better in the detection of EN2 in prostate cancer cells, and it was found that this antibody can be used to detect antigens under non denaturation conditions .

Example 8. Antibody Sensitivity Evaluation of Recombinant EN2 Protein-ELISA < RTI ID = 0.0 >

Recombinant EN2 protein (antigen for detection) prepared in Example 1-1 was added to a 96-well plate overnight at 4 ° C and blocked with TBST solution containing 2% skim milk at 37 ° C. At this time, the recombinant EN2 protein (antigen for detection) was serially diluted with 0, 0.312ng / well, 0.625ng / well, 1.25ng / well, 2.5ng / well and 5ng / well10ng / well. Antibody Anti-EN2 (mono) was diluted to 3.3 nM. Then, the primary antibody was treated and reacted in each well, and the anti-mouse IgG conjugated with HRP as a secondary antibody was diluted to 1/2000. After the secondary antibody reaction, the cells were washed with TMB (3,3 ', 5,5'-Tetramethylbenzidine) solution and developed with 1 N sulfuric acid. The results obtained from the ELISA reader are shown in Table 1, and the results for Anti-EN2 (mono) are shown in FIG. 7A as a standard line.

Condition ELISA for the concentration of EN2 recombinant protein for detection (ng / ml) value 0 ng / ml 0.312 ng / ml 0.625 ng / ml 1.25 ng / ml 2.5 ng / ml 5 ng / ml 10 ng / ml EN2 (mono) antibody 0.068 0.071 0.088 0.117 0.163 0.295 0.589

According to Table 1 and FIG. 7A, the sensitivity of the anti-EN2 (mono) monoclonal antibody is excellent.

In addition, 10000 g of urine sample was centrifuged for 10 minutes, and the supernatant was collected using an anti-EN2 (mono) monoclonal antibody. 7B) using EN2 protein concentration (using healthy male urine as a negative control (N or Normal)), and EN2 protein concentration in the actual sample (urine) as shown in FIG. 7B We can confirm that there is a difference in EN2 protein concentration in the benign prostatic hyperplasia (P1), prostatitis patient (P2) or prostate cancer patient (P3 ~ P8) specimens.

Therefore, through these results, it is possible to quantitatively analyze the correlation between EN2 detection and cancer progression in the urine as well as qualitative test to determine whether the antibody of the present invention is merely cancer, .

&Lt; Example 9: Antibody sensitivity evaluation on urine sample of prostate cancer patient-Western blot &

Using the western blot, the antibody of the present invention was used to confirm whether the classification of prostate disease patients was confirmed.

To this end, 10000 g of a urine sample of benign prostatic hyperplasia (P1), prostatitis patient (P2) or prostate cancer patient (P3 to P8) used in Example 8 was centrifuged for 10 minutes, and only the supernatant was collected, 15 μl of the supernatant was mixed with 5 μl of 4 × sample buffer, boiled at 100 ° C. for 5 minutes, and used as a Western blotting sample. The concentration of the monoclonal antibody Anti-EN2 (mono) used here was 3.3 nM, and healthy male urine was used as a negative control (N or Normal), and Western blotting was performed by the method of Experimental Example 2. The results are shown in Fig.

Referring to FIG. 8A, it can be seen that the monoclonal antibody of the present invention is an antibody capable of selectively recognizing a urine sample of a prostate cancer patient even through Western blotting.

On the other hand, when a patient sample containing prostatitis and hyperplasia was tested using the same specimen by ELISA for confirmation of PSA, it was found that the diagnosis of prostate cancer patient is not good (see FIG. 8B).

&Lt; Example 10: Sequence analysis and epitope analysis of monoclonal antibody >

Monoclonal cells producing anti-EN2 (mono) antibody were provided to Y biologics (Daejeon), and amino acid sequence analysis of the light and heavy chains of the monoclonal antibody was performed. The results are shown in Fig. SEQ ID NOS: 2 and 3 represent the variable heavy chain amino acid sequence of the antibody, and SEQ ID NOS: 4 to 7 represent the variable light chain amino acid sequence of the antibody, representing the amino acid sequence of the light chain and the heavy chain.

The sequence of the protein used as the antigen and the monoclonal antibody were provided to the Aptlon (Seoul) and subjected to epitope mapping. The results are shown in FIG. 10, which confirms that the amino acid sequence of the epitope is DRPSSGPRSRKPKKK. Monoclonal cells producing the anti-EN2 (mono) antibody thus identified were named EN2-P2-9 and deposited on June 28, 2017 with accession number KCLRF-BP-00405 in the Korean Cell Line Research Foundation.

Korea Cell Line Research Foundation KCLRF-BP-00405 20170628

&Lt; 110 > Moogene Medi Co., Ltd. <120> Specific monoclonal antibody from a specific antigen to EN2          protein or composition of the same for diagnosis          prostate cancer <130> 153 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 333 <212> PRT <213> homo sapiens <400> 1 Met Glu Glu Asn Asp Pro Lys Pro Gly Glu Ala Ala Ala Ala Val Glu   1 5 10 15 Gly Gln Arg Gln Pro Glu Ser Ser Pro Gly Gly Gly Ser Gly Gly Gly              20 25 30 Gly Gly Ser Ser Gly Glu Ala Asp Thr Gly Arg Arg Ala Leu          35 40 45 Met Leu Pro Ala Val Leu Gln Ala Pro Gly Asn His Gln His Pro His      50 55 60 Arg Ile Thr Asn Phe Phe Ile Asp Asn Ile Leu Arg Pro Glu Phe Gly  65 70 75 80 Arg Arg Lys Asp Ala Gly Thr Cys Cys Ala Gly Ala Gly Gly Gly Arg                  85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Aly Gly Gly Gly             100 105 110 Gly Gly Ala Gly Gly Ser Glu Glu Leu Leu Gly Ser Gly Ser Glu         115 120 125 Pro Arg Gln Asn Pro Pro Cys Ala Pro Gly Ala Gly Gly Pro Leu Pro     130 135 140 Ala Ala Gly Ser Asp Ser Pro Gly Asp Gly Gly Gly Gly Ser Lys Thr 145 150 155 160 Leu Ser Leu His Gly Gly Ala Lys Lys Gly Gly Asp Pro Gly Gly Pro                 165 170 175 Leu Asp Gly Ser Leu Lys Ala Arg Gly Leu Gly Gly Gly Asp Leu Ser             180 185 190 Val Ser Ser Asp Ser Ser Ser Ser Gln Ala Gly Ala Asn Leu Gly Ala         195 200 205 Gln Pro Met Leu Trp Pro Ala Trp Val Tyr Cys Thr Arg Tyr Ser Asp     210 215 220 Arg Pro Ser Ser Gly Pro Arg Arg Ser Lys Pro Lys Lys Lys Asn Pro 225 230 235 240 Asn Lys Glu Asp Lys Arg Pro Arg Thr Ala Phe Thr Ala Glu Gln Leu                 245 250 255 Gln Arg Leu Lys Ala Glu Phe Gln Thr Asn Arg Tyr Leu Thr Glu Gln             260 265 270 Arg Arg Gln Ser Leu Ala Gln Glu Leu Ser Leu Asn Glu Ser Gln Ile         275 280 285 Lys Ile Trp Phe Gln Asn Lys Arg Ala Lys Ile Lys Lys Ala Thr Gly     290 295 300 Asn Lys Asn Thr Leu Ala Val His Leu Met Ala Gln Gly Leu Tyr Asn 305 310 315 320 His Ser Thr Ala Lys Glu Gly Lys Ser Asp Ser Glu                 325 330 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VH-antibody-EN2 <400> 2 Gln Val Gln Leu Lys Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Thr   1 5 10 15 Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Tyr              20 25 30 Tyr Phe His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile          35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Glu Cys Ala Pro Lys Phe      50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Lys Thr Ala Tyr  65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Asn Asp Asp Ile Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser             100 105 110 <210> 3 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VH-antibody-EN2 <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Thr   1 5 10 15 Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Tyr              20 25 30 Tyr Phe His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile          35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Glu Cys Ala Pro Lys Phe      50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Lys Thr Ala Tyr  65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Asn Asp Asp Ile Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser             100 105 110 <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-antibody-EN2 <400> 4 Asp Ile Val Met Thr Gln Ala Pro Leu Thr Leu Ser Val Thr Ile Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Val Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 5 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-antibody-EN2 <400> 5 Asp Ile Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Val Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-antibody-EN2 <400> 6 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Val Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-antibody-EN2 <400> 7 Asp Xaa Ile Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly   1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser              20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser          35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Val Asp Ser Gly Val Pro      50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly                  85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 8 <211> 390 <212> PRT <213> Artificial Sequence <220> <223> recombinant EN2 <400> 8 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg              20 25 30 Asp Pro Val Arg Arg Ser Ala Ala Ala Ila Ala Met Glu Glu Asn          35 40 45 Asp Pro Lys Pro Gly Glu Ala Ala Ala Val Glu Gly Gln Arg Gln      50 55 60 Pro Glu Ser Ser Pro Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Ser  65 70 75 80 Pro Gly Glu Ala Asp Thr Gly Arg Arg Ala Leu Met Leu Pro Ala                  85 90 95 Val Leu Gln Ala Pro Gly Asn His Gln His Pro His Arg Ile Thr Asn             100 105 110 Phe Phe Ile Asp Asn Ile Leu Arg Pro Glu Phe Gly Arg Arg Lys Asp         115 120 125 Ala Gly Thr Cys Cys Ala Gly Ala Gly Gly Gly     130 135 140 Gly Gly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly 145 150 155 160 Gly Ser Glu Gln Leu Leu Gly Ser Gly Ser Arg Glu Pro Arg Gln Asn                 165 170 175 Pro Pro Cys Ala Pro Gly Ala Gly Gly Pro Leu Pro Ala Ala Gly Ser             180 185 190 Asp Ser Pro Gly Asp Gly Gly Gly Gly Ser Lys Thr Leu Ser Leu His         195 200 205 Gly Gly Ala Lys Lys Gly Gly Asp Pro Gly Gly Pro Leu Asp Gly Ser     210 215 220 Leu Lys Ala Arg Gly Leu Gly Gly Gly Asp Leu Ser Val Ser Ser Asp 225 230 235 240 Ser Asp Ser Ser Gln Ala Gly Ala Asn Leu Gly Ala Gln Pro Met Leu                 245 250 255 Trp Pro Ala Trp Val Tyr Cys Thr Arg Tyr Ser Asp Arg Pro Ser Ser             260 265 270 Gly Pro Arg Arg Ser Lys Pro Lys Lys Lys Asn Pro Asn Lys Glu Asp         275 280 285 Lys Arg Pro Arg Thr Ala Phe Thr Ala Glu Gln Leu Gln Arg Leu Lys     290 295 300 Ala Glu Phe Gln Thr Asn Arg Tyr Leu Thr Glu Gln Arg Arg Gln Ser 305 310 315 320 Leu Ala Gln Glu Leu Ser Leu Asn Glu Ser Gln Ile Lys Ile Trp Phe                 325 330 335 Gln Asn Lys Arg Ala Lys Ile Lys Lys Ala Thr Gly Asn Lys Asn Thr             340 345 350 Leu Ala Val His Leu Met Ala Gln Gly Leu Tyr Asn His Ser Thr Thr         355 360 365 Ala Lys Glu Gly Lys Ser Asp Ser Glu Thr Arg Thr Arg Pro Leu Glu     370 375 380 His His His His His 385 390 <210> 9 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EN2 peptide - antigen <400> 9 Cys Thr Arg Tyr Ser Asp Arg Pro Ser Ser Gly Pro Arg Ser Ser Lys   1 5 10 15 Pro Lys Lys Lys Asn Pro Asn Lys Glu Asp Lys Arg Pro Arg              20 25 30 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> epitope of EN2 peptide <400> 10 Asp Arg Pro Ser Ser Gly Pro Arg Ser Ser Lys Pro Lys Lys Lys   1 5 10 15

Claims (7)

A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 or 3; And a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, 5, 6 or 7. [ The method according to claim 1,
Wherein said antibody recognizes an EN2 (Engrailed-2, Accession No. NP - 001044.2) protein consisting of the amino acid sequence of SEQ ID NO: 1 or a recombinant protein thereof. The method according to claim 1,
Wherein said antibody is an immunoglobulin G type. A composition for diagnosing the presence or absence of EN2 (Engrailed-2) protein, which comprises an antibody according to any one of claims 1 to 3. A diagnostic agent for prostate cancer, which comprises an antibody according to any one of claims 1 to 3. Hybridoma cell line of accession number KCLRF-BP-00405. A monoclonal antibody characterized by being produced by the hybridoma cell line of claim 6.

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