WO2015137686A1 - Monoclonal antibody ca27 capable of recognizing mycoplasma-infected circulating tumor cell, and detection method using same - Google Patents

Abstract

The present invention relates to a monoclonal antibody CA27 capable of recognizing a Mycoplasma-infected circulating tumor cell, and a method for detecting a Mycoplasma infected-circulating tumor cell using the same. Since the monoclonal antibody CA27 specifically recognizes Mycoplasma p37 protein, it is possible to identify whether or not the Mycoplasma-infected circulating tumor cell is present in the blood of cancer patients. Therefore, the present invention has an advantage of being capable of predicting early cancer metastasis potential in the treatment of cancer patients.

Description

Monoclonal antibody CA27 which can recognize mycoplasma infected blood cancer cells and detection method using the same

The present invention relates to a monoclonal antibody CA27 capable of recognizing mycoplasma infected blood cancer cells and a method for detecting mycoplasma infected blood cancer cells using the same.

Persistent infection of bacteria is known to cause many cancers. One bacterium that is drawing attention for carcinogenicity is mycoplasma, which is the smallest bacterium capable of independent replication. Mycoplasma is a polymorphic, cell-walled prokaryote that adheres to, or enters, eukaryotic membranes and survives well. In particular, mycoplasma grows well in eukaryotes without noticeable symptoms, so mycoplasma infection in animal cell cultures is an important concern.

Mycoplasma hyorhinis is known as a common bacterium in the respiratory tract of pigs. Mycoplasma hyorhinis encoding protein p37 is found in many mycoplasmas as part of a high affinity transport system.

Recent studies have shown that continuous exposure to mycoplasma, such as Mycoplasma Hyorainis, causes the oncogene modifications required for human cancer.

In addition, other studies show that the p37 protein alone is sufficient to promote the invasion and metastasis of cancer cells, suggesting that it is more likely that cancer metastasis will increase with mycoplasma infection.

Circulating tumor cells (CTCs) are cancer cells that migrate through the blood through an epithelial-mesenchymal transition (ETM) from primary epithelial cancer cells to mobile mesenchymal cancer cells (EMT). Chaffer, CL and Weinberg, RA Science 331: 1559-1564,2011). The number of blood cancer cells in peripheral blood is attracting attention because of the poorer prognosis of patients with metastatic cancer. Recently, the markers of metastatic cancer cells have been detected by detecting the cancer cells found in peripheral blood of cancer patients and suggesting the association with cancer prognosis. A method that can be used has been developed. However, the number of cancer cells in the blood is so small that it is not easy to actually separate them from the blood efficiently. In addition, it is difficult to accurately detect and isolate various and very small numbers of CTCs because it is estimated that different CTCs will migrate out of various cancers.

As such, due to the scarcity of blood cancer cells and the absence of specific markers of blood cancer cells in peripheral blood, a method for easily detecting blood cancer cells has not been developed yet.

In order to solve the above problems, the present invention is to provide a novel antibody that can recognize mycoplasma infected blood cancer cells.

Another object of the present invention is to provide a method for detecting mycoplasma infected blood cancer cells using a novel antibody that can recognize mycoplasma infected blood cancer cells.

In order to achieve the above object, the present invention provides a monoclonal antibody CA27 that can recognize mycoplasma infected blood cancer cells.

The monoclonal antibody has IgG3 and κ chains and recognizes mycoplasma p37 protein.

The monoclonal antibody comprises a heavy chain variable region (V _H ) having an amino acid sequence of SEQ ID NO: 1 and a light chain variable region (V _L ) having an amino acid sequence of SEQ ID NO: 2.

The present invention also provides a mycoplasma infected blood cancer cell-specific monoclonal antibody CA27 cDNA encoding the monoclonal antibody CA27.

The monoclonal antibody CA27 cDNA comprises a nucleotide sequence of SEQ ID NO: 3 encoding a heavy chain variable region (V _H ) and a nucleotide sequence of SEQ ID NO: 4 encoding a light chain variable region (V _L ).

The present invention also provides a composition for detecting mycoplasma infected blood cancer cells, including the monoclonal antibody CA27.

The present invention also provides a detection kit for mycoplasma infected blood cancer cells, comprising the monoclonal antibody CA27.

In addition, the present invention comprises the steps of adding a monoclonal antibody CA27 to the cell population; And isolating cells that bind to the monoclonal antibody CA27; It provides a method for separating mycoplasma infected blood cancer cells comprising a.

The invention also encompasses hybridomas that produce monoclonal antibody CA27 or antigen binding sites thereof, comprising B cells fused to immortalized cells obtained from a non-human animal having a genome comprising a heavy chain transgene and a light chain transgene. To provide.

The present invention also provides a method for providing information for detecting mycoplasma infected blood cancer cells, comprising detecting mycoplasma p37 protein using monoclonal antibody CA27 in a sample.

Since monoclonal antibody CA27 of the present invention specifically recognizes mycoplasma p37 protein, it is possible to confirm the presence of mycoplasma-infected cancer cells in the blood of cancer patients. This enables early prediction of the possibility of cancer metastasis in the treatment of cancer patients, thus suggesting a treatment direction that precedes mycoplasma treatment for the treatment of metastatic cancer patients.

1 shows the results of Western blot analysis of epithelial-mesenchymal transition (EMT) related markers in H358, A549 and H1703 cells.

FIG. 2 shows that CA27 recognizes about 40 kDa surface protein in A549 and HepG2 cells, A is the flow cytometry analysis of the CA27 antigen, and B is the Western blot analysis of the CA27 antigen.

3 is a mass spectrum result of CA27 antigen immunoprecipitated by CA27.

Figure 4 shows that the CA27 antigen on A549 and HepG2 cells originated from mycoplasma, A is the result of flow cytometry analysis of A549 cells treated with CA27, and B is not treated after immunoprecipitation by CA27. Western blot analysis of CA27 antigen in A549 and BM-cyclin treated A549 cells.

Figure 5 shows that the CA27 antigen on A549 and HepG2 cells originated from mycoplasma, where A is the result of mycoplasma DNA detection in A549 cells by a commercial polymerase chain reaction (PCR) detection kit. Mycoplasma DNA detection results in HepG2 cells by a commercial polymerase chain reaction (PCR) detection kit.

FIG. 6 shows that CA27 positive cells are red and CD45 positive cells are green as a result of detecting CA27 antigen by immunocytochemical method in human peripheral blood mononuclear cells (PBMC) of liver cancer patients.

FIG. 7 shows the CA27 antigen as a result of detecting CA27 antigen by immunocytochemical method after separation of blood cancer cells (CTC) using biotinylated CA27 and Dynabeads FlowCompTM Flexi kit in liver cancer patient PBMC. Positive cells show red (red arrow) and CD45 positive cells show green (green arrow). Black arrows show remaining magnetic beads.

Figure 8 synthesizes the antibody gene heavy chain (Heavy chain, HC) and light chain (Light chain, LC) variable region cDNA from CA27 hybridoma mRNA and then amplified by PCR the heavy and light chain variable region region using the corresponding primers The result is.

FIG. 9 shows the nucleotide sequence and amino acid sequence of the CA27 antibody heavy chain gene variable region (V _H ), showing the complementarity determining region (CDR) binding to the antigen and residue positions of important amino acids.

FIG. 10 shows the nucleotide sequence and amino acid sequence of the CA27 antibody light chain gene variable region (V _L ), showing the complementarity determining region (CDR) binding to the antigen and residue positions of important amino acids.

Hereinafter, the present invention will be described in more detail.

The present inventors have developed a monoclonal antibody CA27 against mycoplasma p37 protein in mycoplasma infected cancer cells during the research and development of a new surface marker of blood cancer cells, and mycoplasma infected blood cancer cells exist in the blood of cancer patients, especially liver cancer patients. It was found for the first time to complete the present invention.

This enables early prediction of cancer metastasis by detecting mycoplasma infected blood cancer cells during the treatment of metastatic cancer patients, thus suggesting a treatment direction that precedes mycoplasma treatment for the treatment of metastatic cancer patients.

In one specific embodiment, the monoclonal antibody CA27 of the present invention can recognize mycoplasma infected blood cancer cells, has IgG3 and κ chains, in particular mycoplasma p37 protein (FIG. 3).

In addition, the monoclonal antibody CA27 includes a heavy chain variable region (V _H ) having an amino acid sequence of SEQ ID NO: 1 and a light chain variable region (V _L ) having an amino acid sequence of SEQ ID NO: 2.

As used herein, the term "monoclonal antibody" refers to a protein molecule that recognizes a single antigenic site (single epitope) and binds specifically thereto. For the purposes of the present invention, the monoclonal antibody of the present invention specifically binds to cell surface molecules of mycoplasma infected blood cancer cells, and thus is a protein molecule that recognizes cell surface molecules of mycoplasma infected blood cancer cells.

The main site of antibodies that recognize specific epitopes of antigens to form antigen-antibody complexes is that the variable regions of the heavy and light chains, particularly the Complementarity Determining Regions (CDRs), contribute to the formation of such complexes. Variable regions of clonal antibodies, particularly chimeric antibodies, humanized antibodies and the like, including CDRs, are included within the scope of the present invention. The present invention also encompasses functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains as long as they have the binding properties as described above. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function and includes Fab, F (ab '), F (ab') ₂ and Fv.

In another aspect, the present invention provides a mycoplasma infected blood cancer cell-specific monoclonal antibody CA27 cDNA encoding monoclonal antibody CA27.

The monoclonal antibody CA27 cDNA includes a nucleotide sequence of SEQ ID NO: 3 encoding a heavy chain variable region (V _H ) and a nucleotide sequence of SEQ ID NO: 4 encoding a light chain variable region (V _L ).

The present invention also provides a composition for detecting mycoplasma infected blood cancer cells, including the monoclonal antibody CA27. The present invention also provides a detection kit for mycoplasma infected blood cancer cells, comprising the monoclonal antibody CA27.

In addition, the present invention comprises the steps of adding a monoclonal antibody CA27 to the cell population; And it provides a method for separating the mycoplasma infected blood cancer cells comprising the step of separating the cells that bind the monoclonal antibody CA27.

The monoclonal antibody CA27 of the present invention is used to specifically detect mycoplasma infected blood cancer cells, in particular mycoplasma p37 protein, via an antigen-antibody complex reaction.

In the case of a composition comprising the monoclonal antibody CA27 of the present invention, it is prepared in a suitable formulation including an acceptable carrier depending on the mode of administration. Suitable formulations for the mode of administration are known in the art. These agents are administered by any suitable method including parenteral, subcutaneous, intraperitoneal, pulmonary, and intralesional administration if necessary for intranasal and local immunosuppressive treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. The composition comprising monoclonal antibody CA27 of the present invention may be administered in a pharmaceutically effective amount for detecting mycoplasma infected blood cancer cells. Typical dosage levels can be optimized using standard clinical techniques.

In addition, the detection kit may include not only the monoclonal antibody CA27 of the present invention but also tools, reagents, and the like generally used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labels capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like.

If the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

Antigen-antibody complex formation includes tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, complement fixation Complement Fixation Assays, FACS, protein chips, and the like, but are not limited thereto.

Labels that enable qualitative or quantitative measurement of antigen-antibody complex formation include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes. . Enzymes that can be used as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholine esterase, and glucose oxy Multidase, Hexokinase and GDPase, RNase, Glucose Oxidase and Luciferase, Phosphofructokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphopyruvate Decarboxylase, β- Latamases and the like, but is not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like.

In another embodiment, the present invention provides hybridomas that produce monoclonal antibody CA27. The present invention provides B cells isolated from transformed or untransformed non-human animals, eg mice, capable of expressing monoclonal antibody CA27 that specifically binds mycoplasma infected blood cancer cells. Preferably, isolated B cells are obtained from transformed or untransformed non-human animals, such as mice, immunized with mycoplasma infected blood cancer cells or agents enriched in the cells. Subsequently, immortalization of the isolated B cells provides a source of monoclonal antibodies against mycoplasma infected blood cancer cells.

Hybridomas secreting monoclonal antibodies can be cultured in large quantities in vitro or in vivo. The monoclonal antibody produced by the hybridoma may be used without purification, but in order to obtain the best results, it is used after being purified with high purity (eg, 95% or more) according to a method well known in the art. It is desirable to. Such purification techniques can be separated from the culture medium or ascites fluid using purification methods such as, for example, gel electrophoresis, dialysis, salt precipitation, chromatography.

In another aspect, the present invention provides a method for providing information for detecting mycoplasma infected blood cancer cells, comprising detecting mycoplasma p37 protein using monoclonal antibody CA27 in a sample. The sample is preferably serum, but is not limited thereto. In addition, the blood cancer cells are preferably liver cancer cells or lung cancer cells, but is not limited thereto.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples. However, the following examples are only for exemplifying the present invention, and the contents of the present invention are not limited to the following examples, which are obvious to those skilled in the art.

Example 1 Characterization of Cancer Cell Lines and Research Ethics

<1-1> Cancer Cell Line Culture

Human lung cancer cell lines (H358, A549, H1703) were purchased from ATCC, USA, and used 10% fetal bovine serum (WelGene) and antibiotic-antimycotic solution (Life Technologies, Seoul, Korea) in RPMI1640 medium (WelGene, Daegu, Korea). And cultured using. Mycoplasma uninfected A549 cells were purchased from Korea Cell Line Bank. Human hepatocellular carcinoma cell lines HepG2 and FO Maeloma cells were purchased from ATCC, USA and cultured using 10% fetal bovine serum (WelGene) and antibiotic-antimycotic solution (Life Technologies) in DMEM medium (WelGene).

<1-2> Research Ethics and Patient Agreement

Human peripheral blood mononuclear cells (PBMC) were isolated using the method described in Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea). Blood samples from liver cancer patients were collected in heparin coated tubes after receiving consent from liver cancer patients who underwent liver cancer resection at Samsung Medical Center in Seoul. All procedures were approved by Seoul Samsung Hospital IRB. It was analyzed at Sejong University. Cancer progression stage analysis used criteria specified by the American Joint Committee on Cancer (AJCC, 2010).

<1-3> Analysis of Cultured Cancer Cell EMT Markers

Based on the literature that lung cancer cells, A549 cells among cancer cell lines, have semi-mesenchymal properties during the epithelial-mesenchymal transition (EMT) process (Thomson, et al., Cancer Res 65: 9455-9462, 2005; Tauler, et al., Cancer Res 70: 7137-7147, 2010), and EMT markers of cancer cell lines H358, A549, H1703 in culture were analyzed by Western blotting.

The cancer cells were washed with PBS (pH7.4) and then removed with 0.05% trypsin and washed with PBS (pH7.4), followed by lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P- 40, 2 µg / ml aprotinin, 100 µg / ml PMSF, 5 µg / ml leupeptin, 1 mM NaF, 1 mM Na ₃ VO ₄ ), and reacted at 4 ° C. for 20 minutes, followed by centrifugation for 40 minutes at 12,000 rpm. After removing the nucleus, the protein solution was prepared by storing at -70 ℃ until use.

The protein solution thus prepared was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting with nitrocellulose membrane. The membrane was blocked for 2 hours at room temperature using 5% skim milk powder, washed three times with PBST [0.05% Tween 20 phosphate buffer (PBS), pH 7.4], and then the western membrane was washed with rabbit anti-E- Anti-E-cadherin, rabbit anti-snail, mouse anti-vimentin, mouse anti-slug, mouse anti-hnRNP A2 / B1 ( All were reacted with Santa Cruz Biotechnology Inc. antibodies for 1 hour at room temperature. After washing with PBST, anti-rabbit or anti-mouse IgG-HRP (1: 4,000; GE healthcare) was further reacted for 1 hour at room temperature according to the primary antibody. Washed three times with PBST and the protein was identified by ECL detection kit (GE healthcare).

As a result, as shown in FIG. 1, H1703 cells express EMT markers vimentin, snail, slug, hnRNPA2 / B1 well, indicating that they are mesenchymal type, and A549 expresses E-cadherin well so that the epithelial type ( Compared to the epithelial type H358, its expression was characterized by a semi-mesenchymal type showing intermediate expression states of H358 and H1703.

Example 2 Preparation of Hybridoma

<2-1> Bait Immune Injection of Cancer Cells

Recent literature has reported that cancer cells have meso-mesenchymal characteristics of the EMT process (Bednarz-Knoll, et al., Cancer Metastasis Rev 31: 673-687,2012). In order to discover the surface markers of blood cancer cells, antibodies specific to the surface of A549 cells showing semi-mesenchymal characteristics were prepared and used. The antibody manufacturing method is a bait using H358 as a bait immunogen. Immunization methods were used (Choi, HS et al., Cell Tissue Res 333: 197-206, 2008). First, cultured H358 cells were removed with trypsin, and 2 × 10 ⁶ cells were injected into the left foot of 6 female Balb / c mice (DBL, Chungbuk, Korea). After 3 days, the same number of H358 and A549 cells were injected 7 times at 3 days intervals into the left and right soles, respectively. On day 21, the immunized mice were dislocated to the cervical spine and transferred to a clean bench, followed by extraction of popliteal lymph nodes in the right hind paw with sterile scissors and tweezers, followed by frosted slide glass. Using popliteal lymph nodes were prepared by grinding single cells.

FO cells (ATCC, Manassas, VA), myeloma cells, were washed with serum-free media, and cell debris was removed using popliteal lymph node cells and FO cells using a 40 μm cell strainer. . After mixing lymph node cells and FO cells in a ratio of 1: 5, wash them with serum-free medium DMEM (Invitrogen, Seoul, Korea), and then use 1ml 50% PEG1500 (polyethylene glycol, BMS, seoul, Korea). Cells were fused. After washing with serum-free medium, the cells were collected by centrifugation, DMEM containing 20% FBS, HAT component (Sigma-Aldrich, Seoul, Korea), 10% hybridoma cloning factor (Bioveris, Gaithersburg) , MD) mixed solution, the fused cells were dispensed 2 × 10 ⁵ cells per well of 96 well plate, incubated in a 5% CO ₂ , 37 ℃ cell incubator, 200 μl of 20 at 3 days intervals Hybridoma formation was induced by switching to DMEM containing% FBS and HAT components.

<2-2> Cloning of Hybridoma

In order to screen clones expressing antibodies in hybridoma supernatants, a sandwich enzyme-linked immunosorbent assay (ELISA) was used. 100 μl of hybridoma culture solution was added to a plate coated with 2 μg / ml of anti-mouse IgG or IgM antibody, and reacted at 37 ° C. for 1 hour, and then horseradish peroxidase (HRP) of anti-mouse IgG or IgM was added. , Sigma-Aldrich) was further reacted with 1 / 5,000 dilution for 1 hour. Wash the plate with PBST, add a substrate solution containing o-phenylenediamine (OPD, Sigma-Aldrich) and H ₂ O ₂ , and measure the absorbance at 492 nm to produce the antibody. Selected.

A variety of hybridomas were selected, subcultured and subcloned continuously to select 75 hybridomas that ensured stability and secreted monoclonal antibody groups. Isotypes of 75 antibodies were determined using the method suggested in Mouse Immunoglobulin Isotyping Kit (BD Biosciences, Seoul, Korea). Among them, the CA27 antibody was an antibody having an IgG3 and a κ chain, and the recognition antigens and characteristics of the antibody were analyzed below.

<2-3> Purification and Biotinylation of Monoclonal Antibody CA27

For pure separation of antibodies, protein G-Sepharose column chromatography was used. After incubating the CA27 hybridomas, approximately 500 ml of the culture was slowly passed through a protein G-Sepharose column (Pharmacia, Sweden) previously equilibrated with PBS (pH 7.4) and the column was passed through a peristatic pump. Connected and washed well with PBS (pH 7.4). After washing was complete, the antibody was eluted with 0.2M Glycin-HCl (pH 2.7). At this time, the eluate was buffered in a tube containing 1M Tris (pH 9.0) prepared in advance. This antibody was used after dialysis in PBS (pH 7.4). Purified antibodies were measured using a BCA protein assay kit (BCA ^™ Protein Assay Kit, Pierce, Rockford, IL) to obtain purified antibodies at a concentration of about 2 mg / ml. Biotinylation of the antibody was performed according to the protocol provided by purchasing the DSB-X-Biotin Protein Labeling Kit (Life Technologies).

Example 3 Binding Specificity Analysis of Monoclonal Antibody CA27

<3-1> Analysis of CA27 Antigen by Flow SightMetric

In order to observe the degree of binding of the antibodies to various cells, Flow Cytometry was performed. Specifically, H358, A549, and HepG2 cancer cells were treated with 0.05% trypsin to remove the cells, washed with PBS (pH 7.4), and then filtered using a 40 μm strainer (BD Biosciences) to separate into single cells. . PBMC and cancer cells 5 x 10 ⁵ cells were mixed in PBA (1% bovine serum albumin, 0.02% NaN ₃ in PBS, pH7.4), and then the CA27 antibody was reacted at 4 ° C for 30 minutes. After washing twice with PBA, the primary antibody and the corresponding anti-mouse IgG-FITC (BD Biosciences) were further reacted at 4 ° C. for 30 minutes. After washing twice with PBA, antibody response was analyzed for propidium iodide (PI) -negative cells using FACS Calibur and Cell Quest software (BD sciences).

As a result, as shown in FIG. 2A, the CA27 antibody binds to A549 and HepG2 without binding to H358 and PBMC.

<3-2> CA27 antigen western analysis

H358, A549, HepG2 cells in culture were washed with PBS (pH7.4) and then lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 μg / ml aprotinin, 100 After reacting for 20 minutes at 4 ° C. using ㎍ / ml PMSF, 5㎍ / ml leupeptin, 1mM NaF, 1mM Na ₃ VO ₄ ), centrifuged at 12,000rpm for 40 minutes to remove nuclei. Protein solution was prepared by storage at ℃. The protein solution thus prepared was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blotting with nitrocellulose membrane. The membrane was blocked for 2 hours at room temperature using a blocking solution containing 5% skim milk powder in PBST, and reacted for 1 hour at room temperature in a solution containing 1 μg of CA27 antibody in PBST. After washing three times with PBST, anti-mouse IgG-HRP (1: 4,000; GE healthcare) was further reacted for 1 hour at room temperature. Washed three times with PBST and the protein was identified by ECL detection kit (GE healthcare). As a result, as shown in FIG. 2B, the CA27 antibody recognizes about 40 kDa protein in HepG2 and A549.

Example 4 Antigen Identification Recognized by CA27

<4-1> Identification of Antigen Precipitated by CA27

SDS gels containing proteins immunoprecipitated by CA27 were stained with Commassie G250 (BIO-RAD) according to the supplier's protocol. Immunoprecipitated 40 kDa protein was cleaved and modified into small fragments using modified porcine trypsin modified according to Shevchenko (Shevchenko, et al., Anal. Chem. 68: 850-858, 1996). Digested.

The gel was washed with 50% acetonitrile to remove impurities such as SDS, organic solvent, and dyeing reagent from the gel pieces. It was then reacted with trypsin (8-10 ng / μl) at 37 ° C. for 8-10 hours. Proteolysis was terminated by the addition of 5 μl of 0.5% trifluoroacetic acid. Protein fragments cut by trypsin were recovered in aqueous solution and desalted and concentrated to 1-5 μl volume using C18ZipTips (Millipore).

This concentrate was mixed with α-cyano-4-hydroxycinnamic acid saturated in the same amount of 50% aqueous acetonitrile and loaded onto the target plate for mass spectrometry. The mass spectrometer used Ettan MALDI-TOF (Amersham Biosciences). Protein fragments loaded on the target plate were vaporized by 337 nm N2 laser irradiation and then accelerated by a 20 Kv injection pulse. Mass spectra for each protein spot were obtained by cumulative peaks of 300 laser shots. For the analysis of the mass spectrum, the ion peak m / z (842.510, 2211.1046) of the peptide generated by the autolysis of trypsin was used as the standard peak. ProFound search engine (http://129.85.19.192/profound_bin/WebProFound.exe) developed by Rockefeller University was used to identify proteins from the mass spectra.

As a result, as shown in FIG. 3, it was confirmed that the antigen protein recognized by CA27 was Mycoplasma hyorhinis p37 protein (FIG. 3). The underlined amino acids in FIG. 3 represent peptides actually identified by mass spectrometry, showing that p37 and 177 amino acids match.

<4-2> Validation of CA27 Antigen

Mycoplasma hyorhinis p37 protein is found in at least 11 different mycoplasma species and shows structural similarity to p37 in other species (Sippel, et al., J Bacteriol 191: 2585-2592.2009). The results of the identification of CA27 antigens indicate that A549 and HepG2 cells used as immunogens were infected with mycoplasma and were the resulting antibodies.

Therefore, in order to check whether A549 in culture is infected with mycoplasma, mycoplasma-free A549 was purchased from the Korean Cell Line Bank and cultured. BM-Cyclin 1 (10) μg / ml) (Roche, Seoul, Korea) was treated for 3 days and again treated with BM-cyclin 2 (5 μg / ml) (Roche) for 4 days. After the same treatment was repeated once more, it was analyzed again by flow cytometry using the CA27 antibody in the same manner as in Example <3-1>.

As a result, as shown in FIG. 4A, CA27 binding was not observed in newly purchased mycoplasma-free-A549, and CA27 binding was significantly decreased in cells treated with BM-Cyclin, unlike cells not treated. .

In addition, in order to compare the immunoprecipitation of antigens recognized by CA27 in B549-treated A549 cells and untreated A549 cells, the cell culture plate was washed three times with PBS (pH 7.4), followed by lysis buffer (25 mM). Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 µg / ml aprotinin, 100 µg / ml PMSF, 5 µg / ml leupeptin, 1 mM NaF, 1 mM Na ₃ VO ₄ ) After 20 minutes of reaction at 4 ℃, centrifuged for 40 minutes at 12,000rpm speed to remove the nucleus and stored at 70 ℃ until the upper cell extract used.

To remove proteins that bind non-specifically to Protein-G-Plus Agarose (Santa Cruz Biotechnology, Santa Cruz, CA), add 20 μl Protein-G-Plus Agarose to the cell extract of about 1 x 10 ⁷ cells. After reacting for 2 hours at ° C., proteins bound to the protein-G-plus agarose were removed by centrifugation, and the remaining supernatant was recovered.

In order to immunoprecipitate the antigen recognized by CA27, 2 μg of the antibody was added to the supernatant and reacted at 4 ° C. for 12 hours, followed by 20 μl Protein-G-Plus Agarose, followed by further reaction at 4 ° C. for 2 hours. . The immunoprecipitated immune mixture was washed 10 times using a lysis solution, 5 × sample buffer was added to elution the antigen bound to the antibody, and boiled at 100 ° C. for 5 minutes. In the same way it was analyzed by Western blotting.

As a result, in A549 treated with BM-Cyclin as shown in FIG. 4B, it was confirmed that 40 kDa CA27 antigen disappeared.

The results indicate that A549 cells are infected with mycoplasma, and thus, 1 million A549 cells in culture were recovered with trypsin and analyzed for DNA detection using a mycoplasma DNA detection kit (iNtRON Biotechnology, Seongnam, Korea). .

As a result, mycoplasma DNA was not detected in A549 cells treated with BM-cyclin but not in A549 cells treated with BM-cyclin (FIG. 5A), and the same result was obtained in HepG2 cells treated with or without BM-cyclin (FIG. 5A). 5B). These results indicate that A549 cells and HepG2 cells used in the experiment were infected with mycoplasma. In addition, the CA27 antibody established using A549 infected with mycoplasma as an immunogen was recognized by recognition of the p37 protein derived from the infected mycoplasma.

Example 5 Analysis of CA27 Antigen in Hepatocellular Carcinoma Patients

<5-1> Analysis of CA27 Antigen in PBMC of Liver Cancer Patients by Immunocytochemical Method

Recently, many literatures have shown that sustained exposure of mycoplasma, such as Mycoplasma hyorhinis , to human cells causes oncogenic transformation (Dennis, et al., Urology 60: 78-83,2002; Pehlivan, et al. , Urology 65: 411-414, 2005; Huang, et al., World J Gastroenterol 7: 266-269,2001; Ning and Shou Ai Zheng 23: 602-604,2004). In addition, the mycoplasma protein p37 alone has been reported to increase the invasiveness and metastasis of cancer cells.

Therefore, we investigated how mycoplasma infection affects the metastasis of cancer cells in liver cancer patients. Blood cancer cells (CTCs) pass through the blood as the primary cancer moves to other organs and can be analyzed by analyzing the blood. PBMC was isolated from blood from healthy adults and three liver cancer patients (hepatocellular carcinoma, HCC) as in Example <1-2>. After preparing a slide coated with poly-L-lysine (0.1 mg / ml), the separated blood was attached by centrifugation at 300 × g for 6 minutes using a cytospin centrifuge, and then 4 ° C. with 4% paraformaldehyde (PFA). Cells were fixed by treatment for 15 minutes at. After washing with PBS (pH 7.4), cells were blocked for 1 hour with blocking solution (10% horse serum, 0.1% BSA, PBS, pH 7.4). After reacting the CA27 antibody for 12 hours at 4 ° C and washing with PBS (pH7.4), light was blocked at room temperature with Dylight649-conjugated anti-mouse IgG (Vector Laboratories, Seoul, Korea) and Alexa488-conjugated CD45 antibody. The reaction was continued for 1 hour. After washing with PBS, nuclei were stained using DAPI (4,6-diamidino-2-phenylindole).

As a result, as shown in FIG. 6, the CA27 antibody did not bind at all in the healthy adult, while the CA27 antibody was found in some cells among the most green cells, which are CD45-positive cells, which are blood-derived cells in the second patient. Was observed to bind in red (Fig. 6). Although detected in only one of the three patients, these results indicate that mycoplasma-infected blood cancer cells are present in liver cancer patients' blood.

<5-2> Separation and Analysis of Blood Cancer Cells of Liver Cancer Patients Using CA27 Antibody

After removing the PBMC when analyzed by CA27 antibodies since many blood leukocytes, so very few of the accurate analysis of the blood cancer cells (CTC) is difficult, the blood cancer cells using Dynabeads FlowComp ^TM Flexi Kit (LifeTechnologies) using CA27 antibody from PBMC Was separated.

First, 100 mycoplasma infected A549 cells were mixed with 1 million blood of healthy persons as a control, and then suspended in 0.5 ml of cold separation solution (PBS, pH 7.4, 0.1% BSA and 2 mM EDTA). In the same manner as in> 10 μg of CA27 antibody biotinylated at 4 ℃ shaking for 30 minutes.

The reacted cells were harvested after adding 1 ml of separation solution, suspended in 1 ml of separation solution, and then added with 10 μl (1.5 × 10 ⁷ beads) of Dynabeads (LifeTechnologies) and shaken at 4 ° C. for 15 minutes. The target cells were recovered by exposure to magnets for 2-3 minutes, and the supernatant containing non-target cells was removed. The complex containing the cells and magnetic beads was washed three times with a separation solution and the target cells were reacted for 10 minutes while shaking with FlowComp Release Buffer (LifeTechnologies) to remove the magnetic beads. The supernatant was carefully recovered and centrifuged at 600 xg for 10 minutes to recover cells and suspended in 200 μl of separation solution.

The recovered cells were attached to poly-L-lysine-coated slides using a cytospin centrifuge in the same manner as in Example <5-1>. The prepared slides were immunocytochemically analyzed with CA27 antibody and CD45 antibody in the same manner as in Example <5-1>, and the CA27 positive and CD45 negative cells were counted and analyzed. As a result, as shown in Table 1, about 22-26 cells were recovered from the 100 cells, indicating a recovery rate of about 24%.

Next, in the blood of four liver cancer patients, blood cancer cells were separated using biotinylated CA27 antibody and Dynabeads FlowComp ^™ Flexi Kit (LifeTechnologies), and analyzed as described above. Observing the blood cancer cells isolated from the patient 6, the cells showed atypical appearance, unlike the round-shaped white blood cells that were not partially removed in the separation process (Fig. 7A, 7B left panel), CA27 positive cells were CD45 negative blood-derived It is different from CD45 positive cells.

When the nuclei of these CA27 positive and CD45 negative cells were stained with DAPI, the nuclei and cytoplasm ratios were larger than those of leukocytes, and also showed atypical nucleus shapes (FIGS. 7A and 7B). This form is characteristic of typical cancer cells (Fehm, T et al., Cytotherapy 7: 171-185,2005) and confirms that isolated CA27 positive cells are blood cancer cells. These CA27 positive and CD45 negative serum cancer cells were detected in patients 5 and 6 of 4 patients (Table 1). These results show for the first time that mycoplasma infected blood cancer cells are present in the blood of liver cancer patients.An important reference to consider for suppressing cancer metastasis in the treatment of metastatic liver cancer patients is to treat mycoplasma first if it is infected with mycoplasma. It suggests that the metastasis can be effectively suppressed.

Table 1 Classification of patients carcinoma Cancer progression stage Total CTC Amount (per 8.5 ml) Separation Method Control normal - 0 Direct Experimental group # 01 HCC pT1 0 Direct # 02 HCC pT2 24 Direct # 03 HCC pT2 0 Direct # 04 HCC pT2 One Dynabead # 05 HCC pT3a 12 Dynabead # 06 HCC pT2 30 Dynabead # 07 HCC pT2 0 Dynabead A549 (100 cells) Cell line spiked 22 Dynabead A549 (100 cells) Cell line spiked 26 Dynabead

Example 6 Monoclonal Antibody CA27 Antibody Gene and Amino Acid Analysis

<6-1> Monoclonal Antibody CA27 Gene Amplification

5 × 10 ⁶ hybridoma CA27 cells were harvested by centrifugation, washed with cold phosphate buffer saline (PBS (pH 7.4)), and shaken vigorously with 1 ml of RNA-iso plus (TAKARA, Japan). Mixed The reaction mixture was allowed to react at room temperature for 5 minutes, 200 μl chloroform was stored in ice for 5 minutes, and then centrifuged at 42,000 ° C. for 15 minutes to recover the supernatant. After adding isopropane corresponding to half of the supernatant, the mixture was left at room temperature for 10 minutes, and then centrifuged at room temperature at a rate of 12,000 xg for 10 minutes to precipitate RNA. RNA was washed with 75% ethanol, water was removed and the RNA precipitate was dried in air. Then dissolved in 50 μl of diethyl pyrocarbonate (Diethyl Pyrocarbonate, Sigma, USA) treated distilled water and then measured A260 to quantify the amount of RNA.

To synthesize cDNA, 500 μl of CA27 RNA 3.5 μl and PrimeScript ™ RT reagent Kit 6.5 μl (TAKARA, Japan) were mixed well, followed by reverse transcription polymerase chain reaction at 37 ° C. for 15 minutes to synthesize cDNA. To clone the CA27 antibody gene, a known polymerase chain primer was modified and used (Wang, et al J. Immunol. Methods 233, 167-177, 2000).

Specifically, in order to perform heavy chain cloning using 50ng of CA27 cDNA, an oligo having 5'-GGA GTC GAC AGG GAC CAA GGG ATA GAC AGA TGG-3 'which is a polymerase chain reaction primer corresponding to an IgG3 constant region 5CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TC-3, 5'-CTT CCG GAA TTC SAR GTN MAG CTG SAG TCW GG-3 ' Phosphorus oligonucleotide was mixed with 20 pmole of each 20 pmole in a mixture of 18 μl of total volume solg ™ e-Taq DNA polymerase (0.25 μl, 10 mM dNTP 1 μl, 10 x e-taq buffer 5 μl (Solgent, South Korea) ) And 32 µl of a mixture of tertiary sterile distilled water were added to prepare a transcription polymerase chain reaction mixture. For light chain cloning, 5'-GGT GTC GAC GGA TAC TAC AGT TGG TGC AGC ATC-3 'oligonucleotide 60 pmole and 5'-CGG primer corresponding to the N-terminus of the kappa chain variable region AAG CTT GAY ATT GTG MTS ACM CAR WCT MCA-3 'and 5-GAC ATT GTG CTG ACC CAA TCT CCA GCT TCT-3, 5-GAC ATT CAG CTG ACC CAG TCT CCA-3 Oligonucleotide 20 pmole 22 µl of the mixed solution was mixed with 0.25 µl solg ™ e-Taq DNA polymerase, 1 µl of 10 mM dNTP, 5 µl of 10 x e-taq buffer (Solgent, South Korea) and tertiary sterile distilled water. Μl was added to prepare a transcription polymerase chain reaction mixture.

For efficient cloning of the polymerase chain reaction product, the light chain was assigned a SalI restriction enzyme site at the 3′-primer end, and the HindIII restriction enzyme site was assigned to the 5-primer. In case of heavy chain, EcoRI was assigned to 5-primer and SalI restriction enzyme site was assigned to 3 ′ primer. Each heavy and light chain transcription polymerase chain reaction mixture was reacted 30 times in 1 minute at 95 ° C, 1 minute at 45 ° C, and 2 minutes at 72 ° C. As a result, amplified DNA was obtained at a position corresponding to about 400 bp in length estimated to be a DNA fragment corresponding to the heavy chain constant region, and about 390 bp in length estimated to be a DNA fragment corresponding to the light chain constant region (FIG. 8).

Example 6-2 Cloning and Sequence Analysis of Monoclonal Antibody 297-D4 Gene

In order to clone the CA27 antibody gene amplified in <Example 6-1>, the heavy chain of the polymerase chain reaction product was first treated with EcoRI and SalI, and the light chain was treated with HindIII and SalI and then developed on a 1% agarose gel. About 400 and 390bp of DNA were isolated by gel extraction kit (Gel extraction kit, Qiagen, USA). PBluescript KS + to be used as a vector to clone the heavy chain gene was treated with EcoRI and SalI, and as a light chain gene cloning vector, pBluescript KS + was treated with HindIII and SalI and separated using a gel extraction kit (Gel extraction kit, Qiagen, USA). The two DNAs were linked with T4 DNA ligase (New England Biolab, USA), and the CaCl ₂ method was used for E. coli DH5α. After transformation, clones with DNA inserts of about 400bp in the heavy chain and E. coli clones with the size of about 390bp in the light chain were selected.

For DNA sequencing of antibody genes, the clones were incubated overnight in 5 ml of LB medium containing 50 μg / ml of ampicillin, followed by alkaline lysis to isolate plasmid DNA. The base sequence of the DNA insert was determined. After changing the nucleotide sequences of the heavy and light chain cDNA to amino acids, each amino acid sequence was analyzed using a Kabat database (Johnson G. and Wu, T. T. Nucleic Acids Res. 29: 205-206, 2001). The numbers on the nucleotide sequences of FIGS. 15 and 16 were determined according to Kabat numbering. Analysis of these amino acid sequences revealed that these immunogenes had complete residues and sequences characteristic of the antibody structure (Figs. 9 and 10). Specifically, among the various immunoglobulin groups, the heavy chain of CA27 was assigned to subgroup II (A). And the light chain belongs to subgroup V.

The CDR residues that recognize the antigen are heavy chain, CDR1 26-37, CDR2 50-66, CDR3 99-109, light chain CDR1 24-34, CDR2 50-56, CDR3 89- It corresponded to 97. The structurally essential disulfide bonds involved cysteines 22 and 96 in the heavy chain and cysteines 23 and 88 in the light chain. Analysis of these genes showed that the heavy and light chain genes were functional.

As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the equivalent scope of the claims to be described.

Claims (13)

Monoclonal antibody CA27 capable of recognizing mycoplasma infected blood cancer cells. The monoclonal antibody CA27 according to claim 1, wherein the monoclonal antibody has an κ chain with IgG3. The monoclonal antibody CA27 of claim 1, wherein the monoclonal antibody recognizes mycoplasma p37 protein. The monoclonal antibody according to claim 1, wherein the monoclonal antibody comprises a heavy chain variable region (V _H ) having an amino acid sequence of SEQ ID NO: 1 and a light chain variable region (V _L ) having an amino acid sequence of SEQ ID NO: 2 Antibody CA27. A monoclonal antibody CA27 cDNA that is specific for mycoplasma infected blood cancer cells encoding the monoclonal antibody CA27 of claim 1. The method according to claim 5, wherein the monoclonal antibody CA27 cDNA comprises a nucleotide sequence of SEQ ID NO: 3 encoding a heavy chain variable region (V _H ) and a nucleotide sequence of SEQ ID NO: 4 coding a light chain variable region (V _L ). Monoclonal antibody CA27 cDNA. A composition for detecting mycoplasma infected blood cancer cells, comprising the monoclonal antibody CA27 of any one of claims 1 to 4. A detection kit for mycoplasma infected blood cancer cells, comprising the monoclonal antibody CA27 of any one of claims 1 to 4. Adding the monoclonal antibody CA27 of any one of claims 1 to 4 to the cell population; And Separating cells that bind to the monoclonal antibody CA27; Mycoplasma infected blood cancer cell separation method comprising a. Claims 1 to 4 comprising a B cell fused to immortalized cells obtained from a non-human animal having a genome comprising a heavy chain transgene and a light chain transgene, and produces the monoclonal antibody CA27 of claim 1 or an antigen binding site thereof. Hybridoma to say. A method for providing information for detecting mycoplasma infected blood cancer cells, comprising detecting a mycoplasma p37 protein using a monoclonal antibody CA27 in a sample. 12. The method of claim 11, wherein said sample is serum. The method of claim 11 or 12, wherein the blood cancer cells are liver cancer cells or lung cancer cells, characterized in that the method for providing information for detecting mycoplasma infected blood cancer cells.

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