KR101630228B1 - Monoclonal antibody CA27 and detecting method using the same - Google Patents

Abstract

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, wherein the monoclonal antibody CA27 specifically recognizes mycoplasma p37 protein, The presence of blood cells infected with mycoplasma in the blood of the patient can be confirmed, so that the possibility of cancer metastasis can be predicted early in the treatment of cancer patients.

Description

Monoclonal antibody CA27 and detecting method using same

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

Continuous infections of bacteria are known to induce many cancers (Oikonomopoulou, K., et al., Clin. Cancer Res. 19: 2834-2841, 2013; Tsai, S., et al., Proc. Sci. USA 92: 10197-10201, 1995). One of the bacteria that is attracting attention as a carcinogen is mycoplasma, and mycoplasma is the smallest bacteria that can reproduce independently. Mycoplasma is a polymorphic prokaryotic cell wall-free organism that attaches to eukaryotic cells or enters cells and survives well. In particular, mycoplasmas grow well in eukaryotes without noticeable symptoms, so mycoplasma infection in animal cell cultures is an important concern.

Mycoplasma hyorhinis is known to be a common bacterial symbiotic organ in the respiratory tract of pigs (Razin, S., et al., Microbiol. Mol. Biol. Rev. 62: 1094-1156, 1998). The Mycoplasma hyorhinis encoding protein p37 has been found in many mycoplasmas as part of a high affinity transport system (Sippel, KH., Et al., J. Bacteriol. 191: 2585-2592, 2009).

Recently, several studies have reported that sustained exposure to mycoplasma, such as mycoplasma enzymolysis, leads to oncogenic genetic alterations necessary for human cancer development (Pehlivan, M. et al., Urology 65: 411-414, 2005; Huang , S. et al., World J. Gastroenterol. 7: 266-269, 2001; Ning, JY., Et al., Ai Zheng 23: 602-604, 2004).

In addition, other studies have shown that p37 protein alone is sufficient to promote the invasion and metastasis of cancer cells (Ketcham, CM, et al., Mol. Cancer Ther. 4: 1031-1038, Cancer Ther. 7: 530-537, 2008). In the case of mycoplasma infection, the cancer metastasis is further increased (see, for example, Gong, M., et al. It is possible to expect that

Circulating tumor cells (CTC) are cancer cells that migrate through the blood through epithelial-mesenchymal transition (EMT), which is a primary epithelial cancer cell (see, for example, Chaffer, CL and Weinberg, RA Science 331: 1559-1564,2011). The number of blood cancer cells in peripheral blood has been attracting attention due to the worse prognosis of metastatic cancer patients. Recently, the detection of blood cancer cells found in peripheral blood of cancer patients has been linked to cancer prognosis, (Nagrath, S et al., Nature 450: 1235-1239, 2007). However, the number of blood cancer cells is so small that it is not easy to effectively isolate blood from the blood. It is also difficult to accurately detect and isolate a wide variety of very few CTCs because it is estimated that different CTCs will migrate out of various cancer types.

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

Korean Patent Publication No. 2013-0014322

In order to solve the above problems, it is an object of the present invention to provide a novel antibody capable of recognizing cancer cells infected with mycoplasma.

It is another object of the present invention to provide a method for detecting mycoplasma-infected blood cancer cells using a novel antibody capable of recognizing cancer cells infected with mycoplasma.

In order to achieve the above object, the present invention provides a monoclonal antibody CA27 capable of recognizing mycoplasma-infected blood cancer cells.

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

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

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

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

In addition, the present invention provides a composition for detecting mycoplasma-infected blood cancer cells, comprising the monoclonal antibody CA27.

In addition, the present invention provides a detection kit for mycoplasma-infected blood cancer cells, which comprises the monoclonal antibody CA27.

The present invention also relates to a method of treating cancer, comprising the steps of: adding monoclonal antibody CA27 to a population of cells; And separating cells that bind to said monoclonal antibody CA27; And a method for isolating mycop eczma-infected blood cancer cells.

The invention also encompasses a hybridoma comprising a B cell fused to an immortalized cell obtained from a non-human animal with a genome comprising a heavy chain transgene and a light chain transgene and comprising a monoclonal antibody CA27 or a hybridoma producing its antigen binding site Lt; / RTI >

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

Since the monoclonal antibody CA27 of the present invention specifically recognizes the mycoplasma p37 protein, the presence of cancer cells infected with mycoplasma in the blood of a cancer patient can be confirmed. This will allow early prediction of cancer metastasis in the treatment of cancer patients, thus suggesting a course of treatment leading to mycoplasma therapy for the treatment of metastatic cancer patients.

Figure 1 shows 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, wherein A is the result of flow cytometric analysis of CA27 antigen and B is the result of Western blot analysis of CA27 antigen.
Figure 3 shows the mass spectral results of CA27 antigen immunoprecipitated by CA27.
FIG. 4 shows that CA27 antigen on A549 and HepG2 cells originate from mycoplasma, wherein A is the result of flow cytometric analysis of A549 cells treated with CA27, B is immune-precipitated by CA27, Western blot analysis of CA27 antigen in A549 and BM-cyclin treated A549 cells.
FIG. 5 shows that CA27 antigen on A549 and HepG2 cells originated from mycoplasma. A is the result of detection of mycoplasma DNA in A549 cells by a commercial polymerase chain reaction (PCR) detection kit, This is the result of detection of mycoplasma DNA in HepG2 cells by a commercial polymerase chain reaction (PCR) detection kit.
FIG. 6 shows the results of detection of CA27 antigen by immunocytochemical method in human peripheral blood mononuclear cells (PBMC) of liver cancer patient. As a result, CA27-positive cells are red and CD45-positive cells are green.
FIG. 7 shows the results of detection of CA27 antigen by immunocytochemistry after separating blood cancer cells (CTC) using biotinylated CA27 and Dynabeads FlowCompTM Flexi kit in liver cancer patient PBMC. As a result, CA27 Positive cells are red (red arrow) and CD45 positive cells are green (green arrow). The black arrow shows the remaining magnetic beads.
FIG. 8 shows the results of synthesizing heavy chain (HC) and light chain (LC) variable region cDNAs in CA27 hybridoma mRNA, amplifying the heavy and light chain variable region using PCR primers Results.
FIG. 9 is a diagram showing the nucleotide sequence and amino acid sequence of the CA27 antibody heavy chain variable region (V _H ), which shows positions of complementary determining regions (CDRs) binding to the antigen and residues 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 ), and shows the position of the complementary determining region (CDR) binding to the antigen and the residue of the important amino acid.

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

The present inventors have been studying a new surface marker for blood cancer cells, and have developed a monoclonal antibody CA27 against mycoplasma p37 protein in mycoplasma-infected cancer cells, and found that cancer cells, especially mycoplasma-infected blood cancer cells, The present inventors have completed the present invention.

Therefore, it is possible to predict the possibility of cancer metastasis by detecting mycoplasma - infected blood cancer cells in the treatment of metastatic cancer patients. Therefore, it is possible to suggest the treatment prior to mycoplasma therapy for the treatment of metastatic cancer patients.

In one specific embodiment, the monoclonal antibody CA27 of the present invention is capable of recognizing mycoplasma infected blood cancer cells and has IgG3 and kappa chains, in particular the mycoplasma p37 protein (Fig. 3).

The monoclonal antibody CA27 also comprises a heavy chain variable region (V _H ) having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (V _L ) having the amino acid sequence of SEQ ID NO: 2.

The term "monoclonal antibody" in the present invention means a protein molecule that recognizes and binds to a single antigenic site (single epitope). For the purpose of the present invention, the monoclonal antibody of the present invention specifically binds to the cell surface molecules of cancer cells infected with mycoplasma, and thus is a protein molecule that recognizes cell surface molecules of cancer cells infected with mycoplasma.

Since the variable region of the heavy chain and light chain, particularly the complementarity determining region (CDR), contributes to the formation of this complex, the main part of the antibody recognizing the specific epitope of the antigen and forming the antigen-antibody complex, A chimeric antibody, a humanized antibody, etc., including a CDR, are included in the scope of the present invention. The present invention also includes 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. The functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') ₂ and Fv.

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

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

In addition, the present invention provides a composition for detecting mycoplasma-infected blood cancer cells, comprising the monoclonal antibody CA27. In addition, the present invention provides a detection kit for mycoplasma-infected blood cancer cells, which comprises the monoclonal antibody CA27.

The present invention also relates to a method of treating cancer, comprising the steps of: adding monoclonal antibody CA27 to a population of cells; And separating the cell that binds to the monoclonal antibody CA27. The present invention also provides a method for isolating cancer cells infected with mycoplasma.

The monoclonal antibody CA27 of the present invention is used for specifically detecting mycoplasma-infected blood cancer cells, particularly mycoplasma p37 protein, through an antigen-antibody complex reaction.

In the case of a composition comprising the monoclonal antibody CA27 of the present invention, an appropriate preparation is prepared containing an acceptable carrier depending on the administration mode. Formulations suitable for the mode of administration are well known in the art. These agents are administered by a suitable method including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, if necessary, for intranasal and local immunosuppressive treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The composition comprising the monoclonal antibody CA27 of the present invention can be administered in a pharmaceutically effective amount to detect mycop eczma-infected blood cancer cells. Typical dosage levels can be optimized using standard clinical techniques.

The detection kit may include the monoclonal antibody CA27 of the present invention as well as tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling substances capable of producing a detectable signal, solubilizers, cleaning agents, buffers, stabilizers, and the like.

When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

Antigen-antibody complex formation may be induced by immunofluorescence staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, Complement Fixation Assay, FACS, protein chip, and the like.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . Enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, A prodrug thereof, a prodrug thereof, a prodrug, a hexose kinase, a GDPase, an RNase, a glucose oxidase and a luciferase, a phosphofructokutase, a phosphoenolpyruvate carboxylase, an aspartate aminotransferase, a phosphoenolpyruvate decarboxylase, Ritamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, _{hydroquinone, K 4 W (CN) 8} , [Os (bpy) 3] 2+ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radiation isotopes 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 and ¹⁸⁶ Re.

In another aspect, the invention provides a hybridoma producing the monoclonal antibody CA27. The present invention provides B cells isolated from transgenic or non-transformed non-human animals, e. G. Mice, capable of expressing the monoclonal antibody CA27 that specifically binds to mycoplasma infected blood cancer cells. Preferably, the isolated B cells are obtained from a non-human animal, such as a mouse, that has been transformed or not transformed with mycoplasma-infected blood cancer cells or the cell-enriched preparation. Followed by immortalization of isolated B cells to provide a source of monoclonal antibodies against mycoplasma infected blood cancer cells.

Hybridomas that secrete monoclonal antibodies can be cultured in vitro or in vivo in large quantities. The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody may be purified and used in high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such purification techniques can be separated from the culture medium or ascites fluid using, for example, purification methods such as gel electrophoresis, dialysis, salt precipitation, chromatography, and the like.

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

Hereinafter, the configuration of the present invention will be described in more detail with reference to examples. It is to be understood, however, that the following examples are illustrative of the invention and are not intended to limit the scope of the invention to those skilled in the art.

< Example  1> Cancer cell line   Character analysis and research ethics

<1-1> Cancer cell line  culture

Human lung cancer cell lines (H358, A549, H1703) were purchased from ATCC, Inc., USA, and 10% fetal bovine serum (WelGene) and antibiotic-antimycotic solution (Life Technologies, Seoul, Korea) were added to RPMI1640 medium (WelGene, Daegu, Korea) Lt; / RTI &gt; Mycoplasma-infected A549 cells were purchased from Korean Cell Line Bank. Human hepatoma cell line HepG2 and FO maeloma cells were purchased from ATCC USA and cultured in DMEM medium (WelGene) using 10% fetal bovine serum (WelGene) and antibiotic-antimycotic solution (Life Technologies).

<1-2> Research Ethics and Patient Consent

Human peripheral blood mononuclear cells (PBMCs) were isolated using the method described by Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea). Blood samples from patients with hepatocellular carcinoma were obtained from hepatocellular carcinoma patients who underwent hepatocarcinoma resection at Seoul Samsung Hospital and collected with heparin coated tubes. All procedures were approved by IRB of Seoul Samsung Hospital. Sejong University analyzed it. The cancer progression phase analysis used criteria specified by the American Joint Committee on Cancer (AJCC, 2010).

&Lt; 1-3 > Cultured cancer cells EMT Marker  analysis

Based on the literature that lung cancer cell A549 cells among cancer cells have semi-mesenchymal characteristics during the epithelial-mesenchymal transition (EMT) process (Thomson, et al. The characteristics of the EMT markers of cultured cancer cell lines H358, A549 and H1703 were analyzed by Western blotting. The results are shown in Table 1.

After the cells were washed with PBS (pH 7.4), the cells were washed with PBS (pH 7.4), and then dissolved in a buffer solution (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P- 40 μg / ml aprotinin, 100 μg / ml PMSF, 5 μg / ml leupeptin, 1 mM NaF, 1 mM Na ₃ VO ₄ ) at 4 ° C. for 20 minutes and then centrifuged at 12,000 rpm for 40 minutes After removing the nuclei, the protein solution was prepared at -70 ° C until use.

The prepared protein solution was separated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blotting with a nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk at room temperature for 2 hours, washed three times with PBST (phosphate buffer (PBS), pH 7.4 with 0.05% Tween 20), and the Western membrane was washed with rabbit anti- Anti-E-cadherin, rabbit anti-snail, mouse anti-vimentin, mouse anti-slug, mouse anti-hnRNP A2 / B1 All from Santa Cruz Biotechnology) were reacted 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 with an ECL detection kit (GE healthcare).

As a result, as shown in FIG. 1, H1703 cells exhibit well-expressed EMT markers vimentin, snail, slug, hnRNPA2 / B1 and are of mesenchymal type. A549 expresses E-cadherin well, Expression of H358 and H1703 showed a semi-mesenchymal type of expression compared to that of H358, an epithelial type.

< Example  2> Hybridoma  Produce

<2-1> Immunization of cancer cell bait

Recent literature has reported that blood cancer cells have a moderate-median nature of progression of EMT (Bednarz-Knoll, et al., Cancer Metastasis Rev 31: 673-687,2012). In order to identify surface markers of cancerous cells in blood, antibodies specific to the surface of A549 cells showing moderate-mesenchymal characteristics were prepared and used. The antibody preparation method used H358 as a bait used as a bait immunogen (Choi, HS et al., Cell Tissue Res 333: 197-206, 2008). First, H358 cells in culture were removed with trypsin, and 2 × 10 ⁶ cells were injected into the left footpad of 6 female Balb / c mice (DBL, Chungbuk, Korea). Three days later, the same number of H358 and A549 cells were injected into the left and right footpad seven times at 3-day intervals. On day 21, immunized mice were removed from the cervical vertebra and transferred to a clean bench. The popliteal lymph node of the right hind paw was removed using sterilized scissors and tweezers, and the frosted slide glass was removed. Were used to grind the ganglion lymph nodes and prepare them as a single cell.

FO cells (ATCC, Manassas, Va.), Myeloma cells, were washed with serum-free medium, and the ganglion cells and FO cells were removed from the cell debris using a 40 μm cell strainer . Lymph node cells and FO cells were mixed at a ratio of 1: 5, washed with serum-free DMEM (Invitrogen, Seoul, Korea), and then stained with 1 ml of 50% PEG 1500 (polyethylene glycol, BMS, seoul, Korea) Cells were fused. (Sigma-Aldrich, Seoul, Korea), 10% hybridoma cloning factor (Bioveris, Gaithersburg, Md.) Containing 20% FBS, , MD), the fused cells were divided into 2 × 10 ⁵ cells per well of a 96-well plate, cultured in a 5% CO ₂ , 37 ° C cell culture incubator, and 200 μl of 20 Hybridoma formation was induced by changing to DMEM containing% FBS and HAT components.

<2-2> Hybrid Cloning

A sandwich enzyme linked immunosorbent assay (ELISA) method was used to select clones expressing the antibody first in the hybridoma supernatant. To the plate coated with anti-mouse IgG or IgM antibody at 2 μg / ml, 100 μl of the hybridoma culture was added, followed by reaction at 37 ° C for 1 hour. Then, anti-mouse IgG or IgM horseradish peroxidase (HRP , Sigma-Aldrich) for 1 hour. The plate was washed with PBST, and a substrate solution containing o-phenylenediamine (OPD, Sigma-Aldrich) and H ₂ O ₂ was added, and the absorbance at 492 nm was measured to obtain a clone producing the antibody Were selected.

Various hybridomas were selected and continued subculturing to select 75 Hybridomas that secreted a monoclonal antibody group with a certain stability. Isotype of the 75 kinds of antibodies was determined by performing the method described in the mouse Immunoglobulin Isotyping Kit (BD Biosciences, Seoul, Korea). Among them, CA27 antibody is an antibody with IgG3 and κ chain, and the recognition antigen and characteristics of antibody are analyzed below.

<2-3> monoclonal antibody CA27 &Lt; / RTI & Biotinization

For pure separation of antibody, it was separated by protein G-Sepharose column chromatography method. After culturing the CA27 hybridoma, approximately 500 ml of the culture was slowly passed through a protein G-Sepharose column (Pharmacia, Sweden) pre-equilibrated with PBS (pH 7.4) and the column was transferred to a peristatic pump And then sufficiently washed with PBS (pH 7.4). After completion of washing, the antibody was eluted with 0.2 M of Glycine-HCl (pH 2.7). At this time, the eluate was buffered in a tube containing 1 M of Tris (pH 9.0) prepared in advance. These antibodies were used after dialysis against PBS (pH 7.4). The amount of purified antibody was measured using a BCA protein assay kit (BCA ^™ Protein Assay Kit, Pierce, Rockford, Ill.) To obtain a purified antibody having a concentration of about 2 mg / ml. Biotinylation of the antibody was performed using the protocol provided by purchasing the DSB-X-Biotin Protein Labeling Kit (Life Technologies).

< Example  3> Monoclonal antibody CA27 Binding specificity

<3-1> Flow Site Mitrie  by CA27  Antigen analysis

Flow cytometry was performed to observe the degree of binding of the antibodies to various cells. 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) . PBMC and 5 x 10 ⁵ cells of cancer cells were mixed with PBA (1% bovine serum albumin, 0.02% NaN ₃ in PBS, pH 7.4) and 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 reaction to propidium iodide (PI) - negative cells was analyzed using FACS Calibur and Cell Quest software (BD sciences).

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

<3-2> CA27  antigen Western  analysis

H358, A549, and HepG2 cells in culture were washed with PBS (pH 7.4), and then lysed in a solution buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P- ㎍ / ml PMSF, 5㎍ / ml leupeptin, 1mM NaF, 1mM Na 3 VO 4) after 20 min at 4 ℃, until ready for use after 40 minutes by centrifugation to remove the nuclei -70 speed 12,000rpm using a Lt; 0 &gt; C to prepare a protein solution. The protein solution thus prepared was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% sodium dodecyl sulfate and subjected to western blotting with nitrocellulose membrane. The membrane was blocked with a blocking solution containing 5% skimmed milk powder at room temperature for 2 hours, and reacted with 1 μg of CA27 antibody in PBST for 1 hour at room temperature. After washing three times with PBST, anti-mouse IgG-HRP (1: 4,000; GE healthcare) was further reacted at room temperature for 1 hour. Washed three times with PBST, and the protein was identified with an ECL detection kit (GE healthcare). As a result, as shown in Fig. 2B, the CA27 antibody recognized about 40 kDa protein in HepG2 and A549.

< Example  4> CA27 Identifying this antigen

<4-1> CA27 Immune by Precipitated  Identification of antigens

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 digested and transformed into small fragments using modified porcine trypsin modified according to the method of Shevchenko (Shevchenko, et al., Anal. Chem. 68: 850-858, 1996) .

The gel pieces were washed with 50% acetonitrile to remove impurities such as SDS, organic solvents, and dyeing reagents. Then, it was reacted with trypsin (8-10 ng / mu l) at 37 DEG C for 8-10 hours. Proteolysis was terminated by addition of 5 [mu] l of 0.5% trifluoroacetic acid. Protein fragments cleaved by trypsin were recovered in aqueous solution and desalted and concentrated to a volume of 1-5 이용 using C18ZipTips (Millipore).

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

As a result, as shown in Fig. 3, the antigen protein recognized by CA27 is Mycoplasma hyorhinis p37 protein (Fig. 3). The underlined amino acid in FIG. 3 actually represents a peptide identified through mass spectrometry, showing that the amino acids of p37 and 177 are identical.

<4-2> CA27  Assay of antigen

Mycoplasma The 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 result of the identification of the CA27 antigen indicates that the A549 and HepG2 cells used as immunogen were infected with mycoplasma and the resultant antibody.

Therefore, in order to confirm whether the cultured A549 was infected with mycoplasma, mycoplasma-free A549 was purchased from the Korean Cell Line Bank and cultured. The A549 cells cultured in this laboratory were treated with mycoplasma remover BM-Cyclin 1 (10 (Roche, Seoul, Korea) for 3 days and then treated with BM-cyclin 2 (5 μg / ml) (Roche) for 4 days. After repeating the same treatment one more time, it was analyzed again by flow cytometry using 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-pre-A549, and in the cells treated with BM-cyclin, CA27 binding was remarkably decreased unlike the untreated cells .

To compare the immunoprecipitations of antigens recognized by CA27 in BM-cyclin treated A549 cells and untreated A549 cells, the cell culture plates were washed three times with PBS (pH 7.4), and then lysed in a lysis buffer (25 mM The cells were treated with 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 incubation at 4 ° C for 20 minutes, the nuclei were removed by centrifugation at 12,000 rpm for 40 minutes and then stored at 70 ° C until the upper layer cell extract was used.

To remove proteins that bind nonspecifically to protein-G-plus agarose (Santa Cruz Biotechnology, Santa Cruz, Calif.), 20 μl protein-G-plus agarose was added to approximately 1 x 10 ⁷ cell extracts Deg.] C for 2 hours, and the proteins bound to the protein-G-plus agarose were removed by centrifugation and the remaining supernatant was recovered.

To immunoprecipitate the antigen recognized by CA27, 2 μg of the antibody was added to the collected supernatant, and reacted at 4 ° C. for 12 hours. Then, 20 μl of protein-G-plus agarose was added and further reacted at 4 ° C. for 2 hours . Immunoprecipitated immune mixture was washed 10 times with a dissolving solution and 5 x sample buffer was added to elute the antibody bound to the antibody and boiled at 100 ° C for 5 minutes. And analyzed by Western blotting in the same manner.

As a result, it was confirmed that the 40 kDa CA27 antigen disappeared in the BM-cyclin-treated A549 as shown in Fig. 4B.

The above results indicate that A549 cells were infected with mycoplasma. Therefore, 1 million A549 cells in culture were recovered as 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 BM-cyclin-treated A549 but in BM-cyclin-treated A549 cells (Fig. 5A) and the same results were obtained in BM-cyclin treated or untreated HepG2 cells 5B). These results indicate that A549 cells and HepG2 cells used in the experiment were infected with mycoplasmas. The CA27 antibody, which was established by using A549 infected with mycoplasma as an immunogen, was recognized by recognizing p37 protein derived from infected mycoplasma.

< Example  5> Liver Cancer Patients Blood Cancer Cells CA27  Antigen analysis

<5-1> Patients with liver cancer PBMC By immunocytochemical methods CA27  Antigen analysis

In many recent literature, Mycoplasma Continuous exposure of mycoplasma, such as hyorhinis , has been shown to induce 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). It is also reported that the mycoplasma protein p37 alone increases the invasiveness and metastasis of cancer cells (Ketcham, et al., Mol Cancer Ther 4: 1031-1038, 2005; Gong, et al., Mol Cancer Ther 7: 530-537, 2008).

Therefore, we investigated the effect of mycoplasma infection on cancer cell metastasis in patients with liver cancer. Blood cancer cells (CTC) can be detected by analyzing blood through the blood when primary cancer moves to other organs. PBMC were isolated from healthy adult and hepatocellular carcinoma (HCC) as in Example <1-2>. The slides coated with poly-L-lysine (0.1 mg / ml) were prepared and centrifuged at 300 xg for 6 minutes using a cytospin centrifuge to separate the slides. The slides were then washed with 4% paraformaldehyde (PFA) For 15 minutes to fix the cells. After washing with PBS (pH 7.4), cells were blocked with blocking solution (10% horse serum, 0.1% BSA, PBS, pH 7.4) for 1 hour. CA27 antibody was reacted at 4 ° C for 12 hours, washed with PBS (pH 7.4), blocked with Dylight 649-conjugated anti-mouse IgG (Vector Laboratories, Seoul, Korea) and Alexa488-conjugated CD45 antibody at room temperature And reacted for 1 hour. After washing with PBS, nuclei were stained with DAPI (4,6-diamidino-2-phenylindole).

As a result, as shown in FIG. 6, it can be seen that CA27 antibody does not bind at all in healthy adults, whereas in most of the green cells, which are CD45-positive cells that are blood-derived cells in patient No. 2, CA27 antibody (Fig. 6). Although only one of the three patients was detected, these results indicate that mycoplasma-infected blood cancer cells are present in the blood of patients with liver cancer.

<5-2> CA27  Isolation and Analysis of Cancer Cells in Patients with Liver Cancer Using Antibodies

After removing the PBMC when analyzed by antibody CA27 because many blood white blood cells, so a few accurate analysis of blood tumor cells (CTC) is difficult, with the Dynabeads ^TM FlowComp CA27 antibody from PBMC Flexi Kit (LifeTechnologies) was used to isolate blood cancer cells.

First, 100 million mycoplasma-infected A549 cells were mixed with one million blood of a healthy person as a control group and suspended in 0.5 ml of a cold separation solution (PBS, pH 7.4, 0.1% BSA and 2 mM EDTA) > Were reacted with 10 μg of biotinylated CA27 antibody at 4 ° C for 30 minutes with shaking.

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

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

Next, four in liver blood biotinylated antibodies and Dynabeads FlowComp ^TM CA27 of patients Flexi Kit (LifeTechnologies) was used to isolate cancer cells in blood and analyzed as described above. CA27-positive cells showed CD45-negative cells (Fig. 7A, Fig. 7B, left panel), unlike the round-shaped white blood cells that were not partially removed during the separation process Lt; RTI ID = 0.0 &gt; CD45 &lt; / RTI &gt;

When the nuclei of the CA27-positive and CD45-negative cells are stained with DAPI, the ratio of the nucleus to the cytoplasm is larger than that of the leukocyte and exhibits an irregular nucleus (FIGS. 7A and 7B). This form is a characteristic of typical cancer cells (Fehm, T et al., Cytotherapy 7: 171-185, 2005), confirming that isolated CA27-positive cells are blood cancer cells. These CA27-positive and CD45-negative blood cancer cells were detected in patients 5 and 6 among 4 patients (Table 1). These results are the first to show that mycoplasma-infected blood cancer cells are present in the blood of patients with liver cancer. As an important reference to be considered in order to suppress cancer metastasis in the treatment of metastatic liver cancer patients, mycoplasma should be treated first It is possible to efficiently suppress the transition.

Patient classification carcinoma Cancer progression stage Total CTC amount
(Per 8.5 ml) How to separate Control group 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 buffered saline (PBS, pH 7.4), 1 ml of RNA-iso plus (TAKARA, Japan) and vigorously shaken Mixed. After incubation at room temperature for 5 minutes, 200 μl of chloroform was added, and the mixture was stored on ice for 5 minutes. The mixture was centrifuged at 4 ° C. for 12 minutes at 12,000 × g to recover the supernatant. After adding isopropane, which corresponds to half of the supernatant, the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged at 12,000 xg for 10 minutes at room temperature to precipitate RNA. The RNA was washed with 75% ethanol, the water was removed, and the RNA precipitate was dried in air. Then, it was dissolved in 50 μl of distilled water treated with diethyl pyrocarbonate (Sigma, USA), and the amount of RNA was determined by measuring A260.

In order to synthesize the cDNA, 3.5 ng of CA27 RNA (500 ng) and 6.5 μl of PrimeScript ™ RT reagent Kit (TAKARA, Japan) were mixed well, and then cDNA was synthesized by reverse transcription polymerase chain reaction at 37 ° C for 15 minutes. In order to clone the CA27 antibody gene, known polymerase chain reaction primers were used after modification (Wang, et al., Immunol. Methods 233, 167-177, 2000).

Specifically, in order to carry out heavy-chain cloning using 50 ng of the cDNA of CA27, the nucleotide sequence 5'-GGA GTC GAC AGG GAC CAA GGG ATA GAC AGA TGG-3 ', which is a polymerase chain reaction primer corresponding to the IgG3 constant region 3, 5'-CTT CCG GAA TTC SAR GTN MAG CTG SAG TCW GG-3 ', which is a primer corresponding to 40 pmole of the nucleotide and the N terminus of the heavy chain variable region of the antibody 5-CTT CCG GAA TTC SAR GTN MAG CTG SAG SAG TC- 20 μl of each oligonucleotide was added to a total volume of 18 μl. To the mixture was added 0.25 μl of DNA polymerase, 10 μl of dNTP, and 5 μl of 10 x e-taq buffer (Solgent, Korea ) And tertiary sterilized distilled water was added to the mixture 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 'primer corresponding to the kappa chain constant region, 60 pmoles of the oligonucleotide and 5'-CGG corresponding to the kappa chain variable region N terminus 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 20 pmole of CCA-3 oligonucleotide, 22 μl of a mixture solution of a mixture of 0.25 μl of a DNA polymerase, 10 μl of 10 mM dNTP, 5 μl of 10 x e-taq buffer (Solgent, Korea) and tertiary sterilized distilled water Was added to make a transcription polymerase chain reaction mixture.

In order to efficiently clone the PCR product, a SalI restriction enzyme site was attached to the 3'-primer end and a HindIII restriction site was assigned to the 5-primer in the light chain. In the case of the heavy chain, EcoRI was assigned to the 5-primer and SalI restriction enzyme site was assigned to the 3 'primer. Each of the heavy chain and light chain transcription polymerase chain reaction mixture was reacted 30 times at 95 ° C for 1 minute, 45 ° C for 1 minute, and 72 ° C for 2 minutes. As a result, amplified DNA was obtained at about 400 bp, which is the length of the DNA fragment corresponding to the heavy chain constant region, and about 390 bp, which is the length of the DNA fragment corresponding to the light chain constant region (FIG. 8).

< Example  6-2> Monoclonal antibody 297- D4  Gene Cloning  And base sequence analysis

In order to clone the CA27 antibody gene amplified in the above <Example 6-1>, the heavy chain of the PCR product was first treated with EcoRI and SalI, the light chain was treated with HindIII and SalI, and developed on 1% agarose gel And the DNA corresponding to about 400 and 390 bp was isolated with a gel extraction kit (Qiagen, USA). PBluescript KS + was used as a vector for cloning the heavy chain gene. EcoRI and SalI were used for the cloning, and pBluescript KS + was digested with HindIII and SalI as a light chain gene cloning vector. Then, the product was separated into gel extraction kit (Qiagen, USA). These two DNAs were ligated with T4 DNA ligase (New England Biolab, USA), and E. coli DH5α was transformed with CaCl ₂ After transformation, Escherichia coli clones with a DNA insert of about 400 bp in the heavy chain and about 390 bp in the light chain were selected.

For the DNA sequencing of the antibody genes, the clones were cultured overnight in 5 ml of LB medium containing 50 μg / ml of ampicillin, and the plasmid DNA was isolated by alkaline lysis method, The nucleotide sequence of the DNA insert was determined. The amino acid sequences of the heavy and light chain cDNAs were changed to amino acids and the amino acid sequences were analyzed using Kabat database (Johnson G. and Wu, T.T. Nucleic Acids Res. 29: 205-206, 2001). The numbers on the nucleotide sequences in Figures 15 and 16 were determined according to Kabat numbering. As a result of analysis of the amino acid sequence, these immune genes are perfectly equipped with residues and sequences characteristic of the antibody structure (FIGS. 9 and 10). Specifically, among the various immunoglobulin groups, the heavy chain of CA27 is subgroup II And the light chain belongs to the subgroup V. [

CDR2 is 50-66, CDR3 is 99-109, and CDR1 is 24-34, CDR2 is 50-56, and CDR3 is 89-99 in light chain. In the case of heavy chain, CDR1 is 26-37, CDR2 is 50-66, 97. Structurally essential disulfide bonds were involved in cysteines 22 and 96 in the case of heavy chains and cysteines 23 and 88 in the case of light chains. Analysis of this gene confirmed that the heavy and light chain genes were functional.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

<110> INDUSTRY-ACADEMIA COOPERATION GROUP OF SEJONG UNIVERSITY <120> Monoclonal antibody CA27 for recognizing mycoplasma          infected-circulating tumor cells          same <130> ADP-2014-0274 <150> KR 10-2014-0027501 <151> 2014-03-10 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 133 <212> PRT <213> Mus musculus <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr              20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Arg Arg Leu Glu Trp Val          35 40 45 Ala Tyr Ile Ser Ser Gly Gly Ser Ser Met Tyr Tyr Ser Asp Thr Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys                  85 90 95 Ala Arg Gly Tyr Gly Ser Arg Asn Trp Phe Phe Asp Val Trp Gly Ala             100 105 110 Gly Thr Thr Thr Val Ser Ser Ser Ala Thr Thr Ser Ser Val Ser Val         115 120 125 Tyr Pro Leu Val Pro     130 <210> 2 <211> 116 <212> PRT <213> Mus musculus <400> 2 Asp Ile Gln Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly   1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile          35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser  65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu                  85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala             100 105 110 Pro Thr Val Ser         115 <210> 3 <211> 399 <212> DNA <213> Mus musculus <400> 3 caagttcagc tgcagcagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cgctttcagt agctatgaca tgtcttgggt tcgccagact 120 ccggagagga ggctggagtg ggtcgcatac attagtagtg gtggtggtag catgtactat 180 ccagacactg tgaagggccg attcaccatc tccagagaca gtgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagggggtac 300 ggtagtagaa actggttctt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 360 gctacaacaa cagccccatc tgtctatccc ttggtccct 399 <210> 4 <211> 348 <212> DNA <213> Mus musculus <400> 4 gacattcagc tgacccagtc tccaaaattc atgtccacat cagtaggaga cagggtcagc 60 gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 120 gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240 gaagacttgg cagagtattt ctgtcagcaa tataacagct atcctctcac gttcggtgct 300 gggaccaagc tggagctgaa acgggctgat gctgcaccaa ctgtatcc 348

Claims (13)

A heavy chain variable region (V _H ) having the amino acid sequence of SEQ ID NO: 1; and a light chain variable region (V _L ) having the amino acid sequence of SEQ ID NO: 2. The monoclonal antibody of claim 1, wherein said monoclonal antibody has IgG3 and kappa chains. The monoclonal antibody CA27 of claim 1, wherein the monoclonal antibody recognizes mycoplasma-infected blood cancer cells. The monoclonal antibody CA27 of claim 1, wherein the monoclonal antibody recognizes a mycoplasma p37 protein. A monoclonal antibody CA27 cDNA encoding the monoclonal antibody CA27 of any one of claims 1 to 4. The monoclonal antibody CA27 cDNA according to claim 5, which comprises a base sequence of SEQ ID NO: 3 encoding a heavy chain variable region (V _H ) and a nucleotide sequence of SEQ ID NO: 4 which codes a light chain variable region (V _L ) Lt; RTI ID = 0.0 &gt; CA27 &lt; / RTI &gt; cDNA. A composition for detecting mycoplasma infection, which comprises the monoclonal antibody CA27 of any one of claims 1 to 4. A kit for detecting mycoplasma infection, which comprises 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 a group of cells isolated in vivo; And
Selecting cells that bind to said monoclonal antibody CA27. Comprising B cells fused to an immortalized cell obtained from a non-human animal having a genome comprising a heavy chain transgene and a light chain transgene and producing the monoclonal antibody CA27 or antigen binding site thereof according to any one of claims 1 to 4 A hybrid horse. A method for providing information for detecting mycoplasma infected cells, comprising the step of detecting mycoplasma p37 protein using the monoclonal antibody CA27 of any one of claims 1 to 4 in a sample. 12. The method of claim 11, wherein the sample is serum. 12. The method of claim 11, wherein the cell is a blood cancer cell.

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