KR100473172B1 - A monoclonal antibody recognizing both human erythropoietin and modified erythropoietin and a hybridoma cell line producing the same - Google Patents

Abstract

The present invention provides a fusion cell of Accession No. KCLRF-BP-00071 that produces a monoclonal antibody that recognizes both human erythropoietin (EPO) protein and modified human erythropoietin protein, and the monoclonal antibody produced thereby. .

Description

Monoclonal antibody recognizing both human erythropoietin and modified erythropoietin and a hybridoma cell line producing the same}

The present invention provides novel fusion cells (monbridoma) that produce a monoclonal antibody that recognizes both human erythropoietin (EPO) protein (hereinafter referred to as "EPO") and modified human erythropoietin protein. It relates to a monoclonal antibody produced.

EPO is a glycoprotein with a molecular weight of 30,000-34,000 Da and is a hematopoietic factor that promotes the production and differentiation of red blood cells. The protein binds to receptors on erythrocyte progenitor cells and begins to act, causing an increase in intracellular calcium ion concentrations, an increase in DNA biosynthesis and stimulation of hemoglobin production. This stimulating factor, EPO, can be used as a therapeutic agent for anemia in kidney disease, anemia in premature infants, anemia due to hypothyroidism, and anemia due to malnutrition.

On the other hand, the clinical use of human natural EPO continues to expand, but the short half-life requires an average of about three times a week, causing inconvenience and high cost. Thus, long-term maintenance of their in vivo activity can further reduce their frequency of use.

Many studies have been conducted to increase and maintain the activity of EPO. The mainstream among them is the use of mutagenesis to replace several amino acids in the EPO gene to achieve that goal. In addition, genetic engineering methods can be used to fuse new amino acids, peptides or protein fragments into EPO molecules to dramatically increase in vivo activity by increasing the sugar chain content of EPO, specifically, sialic acid.

 The inventors carried out ongoing studies to develop new methods that can enhance their in vivo activity by increasing the sugar chain content of EPO by fusing new amino acid, peptide or protein fragments into the EPO molecule. As a result, the modified EPO protein obtained by fusing a specific peptide fragment to the carboxy terminus of EPO contains a large amount of amino acids that increase the glycosylation site without affecting the intrinsic activity of the EPO itself, thereby dramatically reducing the half-life in vivo. The findings were found to increase and research was conducted to develop them into pharmaceuticals.

In the present invention, when a monoclonal antibody that recognizes both EPO and modified EPO protein is present, it can be used for purification using affinity column for EPO and modified EPO protein and various immunological studies. It can be useful for quantitative analysis. However, conventionally known monoclonal antibodies against human EPO can only effectively recognize human EPO proteins, but also human EPO analogs or modified proteins with increased sugar content, such as peptides added to the carboxy terminus of human EPO proteins (e.g., Aranesp ™ cancer drug), such as a modified human EPO protein has a problem that does not recognize effectively.

Antibodies usually produced in vivo by antigen stimulation are mixtures of antibodies (polyclonal antibodies) that have various binding properties to the antigen, but some antibody-producing cells produce only one type of antibody. Monoclonal antibody refers to a homogeneous antibody having the same structure formed by cells (monolone) that have grown from a single antibody producing cell. A method for preparing cells that produce monoclonal antibodies was developed in 1975 by Kohler and Milstein. This method involves fusion of B-lymphocytes obtained from the spleen or lymph nodes of an animal immunized with a particular antigen with myeloma cells, which are normal cells with the ability to produce monoclonal antibodies and myeloma cells become cancerous. As cells obtained from B-lymphocytes, which do not have the ability to produce immunoglobulins but have abnormal proliferative capacity to continue growing, consequently, the fusion cells of these cells can produce monoclonal antibodies of B-lymphocytes. The ability to grow and to continue to grow without killing myeloma cells.

When cell fusion is completed, the fusion cells, non-fusion splenocytes, and myeloma cells are mixed in the medium in which the fusion is performed. Therefore, only fusion cells need to be selected. Cells are grown in HAT medium for this purpose. Purine (adenine, guanine) is biosynthesized through two pathways, the endogenous pathway and the salvage pathway, so wild pathogens are not lethal if they lose either pathway. HAT medium contains hypoxanthine, aminopterin and thymidine, which inhibit the endogenes pathway's action by blocking the endogenes pathway's action of dihydrofolate reductase (DHFR). Therefore, non-fused myeloma cells that lack the salvage pathway's hypoxanthine phosphoribosyl transferase (HGPRT) (and thus are resistant to toxic base analogs (thioguanine or azaguuanine)) are also wild. Unlike the strain, the HAT medium does not grow but dies, and non-fused splenocytes die naturally after 1 to 2 weeks after separation, so that only the fusion cells grow in the medium, thereby allowing selection of the fused cells.

However, in spite of the method for producing fusion cells producing such antibodies, no monoclonal antibody that recognizes both human EPO protein and modified human EPO protein has been reported.

Accordingly, the present inventors have conducted continuous research to obtain monoclonal antibodies that recognize both EPO and modified EPO proteins. As a result, the present invention has fused cells prepared by fusion of splenocytes and myeloma cells of mice immunized with EPO protein. The present invention was completed by successfully obtaining monoclonal antibodies by selecting and culturing fusion cells that recognize all of them using EPO and modified EPO.

Accordingly, it is an object of the present invention to provide novel fusion cells that produce monoclonal antibodies that recognize both EPO and modified EPO proteins.

Still another object of the present invention is to provide a monoclonal antibody produced by the fusion cell.

The present invention provides a fusion cell that produces a monoclonal antibody that recognizes both human EPO protein and modified EPO protein. The fusion cell is preferably a fusion cell of Accession No. KCLRF-BP-00071.

The present invention also provides a monoclonal antibody produced by the fusion cells and recognizes both human EPO and modified human EPO proteins.

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

In the present invention, mice are immunized twice with human EPO protein over a period of time in order to produce fusion cells producing monoclonal antibodies that specifically bind to human EPO protein. Human EPO protein may be one produced by genetic recombination methods.

Splenocytes were taken from immunized mice and mixed with 8-azaguanine resistant plasma cell NS-O (Galfre, G and Milstein, C. Methods Enzymol. 73, p3, 1981) in 50% polyethylene glycol (PEG) solution and 50 Cell fusion reaction is carried out by treatment with% PEG and 20% DMSO (dimethyl sulfoxide) solution. PEG acts to enhance intercellular fusion.

After completion of the cell fusion reaction, cells were cultivated in DMEM medium containing HAT, and only fusion cells were selected. After separation and incubation, each cell was isolated to determine whether or not an antibody recognizes both human EPO and modified EPO protein. Cells producing monoclonal antibodies at high titer were isolated by irradiation using ELISA method, and then only antibody-producing cells were isolated using subcloning method by soft agarose. Among them, fusion cells whose antibody production stability has been confirmed for at least 6 months are isolated.

The inventors of the present invention deposited the fusion cells isolated as described above on November 8, 2002 to the Korean Cell Line Research Foundation, a medical institution affiliated with Seoul National University, which is an international depository institution under the Budapest Treaty. BP-00071 has been granted.

Monoclonal antibodies produced from these fusion cells were obtained by culturing the Accession No. KCLRF-BP-00071 fusion cells obtained above in a suitable medium and separating them from the medium by ammonium sulfate precipitation and column chromatography. do. Monoclonal antibodies obtained according to the present invention are attached to a column and used for the purification of human EPO and modified human EPO proteins by chromatography, or used for Western blot, ELISA, etc. And useful for purification.

Example

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to Examples, but this is not intended to limit the scope of the present invention in any way.

Example 1:  Preparation of Fusion Cell No. KCLRF-BP-00071

Mice were first immunized with human EPO protein (CJ Co., Ltd.) produced and purified from CHO cells using a genetic recombination method, and a second immunization was performed one month thereafter, and again three days later, about 10 ³ Splenocytes were harvested and washed with DMEM medium. In addition, about 3.8 × 10 ⁷ plasma cells NS-O showing 8-azaguanine resistance were taken and washed with the same medium. After these two cells were mixed together, the mixture was treated with 0.5 ml of 50% PEG solution (pH 9.0) for 30 seconds, and then treated with 50% PEG and 20% DMSO solution for 30 seconds to perform cell fusion reaction.

Cells were then washed three times with DMEM medium and added to 96-well plates to 2.5 × 10 ⁴ cells per well. At this time, DMEM containing a certain amount of HAT (hypoxanthine 10 ^-4 M, aminopterin 6 x 10 ^-7 M, thymidine 1.6 x 10 ^-6 M) and 5% calf serum was used as a culture medium. Two weeks after the cell fusion reaction, the presence of a monoclonal antibody against human EPO protein was examined in the culture medium in each well. An ELISA method was used to detect very small amounts of antibodies. By the above method, about 1,000 fusion cells were obtained, from which fusion cells producing high titer of monoclonal antibody against EPO protein were isolated.

Using the soft agarose method as a subcloning method, antibody producing cells were quickly isolated from the wells in which the clones grew. That is, 0.8% agarose was added to the culture solution containing HAT, and primary clones were added thereto and cultured for 2 weeks. Two weeks later, 20 randomly selected cells were selected to confirm the production of monoclonal antibodies against human EPO protein. Among the fusion cells, 70 fusion cells were confirmed to obtain antibody production stability over 6 months.

Among the 70 fusion cells thus obtained, human chorionic gonadotropin (HCG) at the carboxy terminus of the present inventors was developed to select a cell line producing a monoclonal antibody that recognizes all modified human EPO proteins in addition to human EPO. ) Modified human EPO protein (CJ Co., Korea Patent Application No. 2001-10-75994) fused to beta subunit carboxy terminal peptide and Aranesp ™, a modified human EPO protein developed by Amgen ELISA tests were performed. This test allowed the selection of cell lines that produce monoclonal antibodies in addition to human EPO that have similar affinities to the modified human EPO protein.

As mentioned above, the present inventors deposited the isolated fusion cells with the Korea Cell Line Research Foundation affiliated with Seoul National University College of Medicine on November 8, 2002, and received accession number KCLRF-BP-00071.

Example 2: Preparation of monoclonal antibodies that specifically bind human EPO and modified human EPO proteins

The fusion cells of Accession No. KCLRF-BP-00071 prepared according to the method of Example 1 were cultured in RPMI1640 medium containing 5% calf serum. The culture solution was collected by 1 L and centrifuged at 10,000 rpm (Sorval) for 10 minutes at 4 ° C to remove cells. The supernatant was adjusted to pH 8 with 2M Tris buffer and ammonium sulfate was added to 50% to precipitate the antibody in solution. The precipitate was collected by centrifugation and dissolved in 50 ml buffer (50 mM Tris-HCl + 100 mM NaCl, pH 7.2) and diafiltration with the same buffer. The dialysis filtrate was passed after equilibrating the DEAE Affi-Gel Blue (Bio Rad) column with 100 ml of the same buffer. The column was also washed with 100 ml of the same buffer and eluted with 70 ml of eluent (50 mM Tris-HCl + 500 mM NaCl, pH 7.2) to obtain the purified antibody protein. The monoclonal antibodies obtained here were used for the purification of the following human EPO and modified human EPO proteins.

Example 3: Preparation of Sepharose 4B Column with Monoclonal Antibody Attached

6 liters of 1 mM hydrochloric acid was poured into Sepharose 4B (Pharmacia), which was activated with 30 g of dried CNBr, and swelled. The gel was washed three times with buffer (0.1 M NaHCO ₃ + 0.5 M NaCl, pH 8.3) and then placed in 150 ml of the same buffer. The monoclonal antibody obtained from the fusion cell of Accession No. KCLRF-BP-00071 was purified and 450 mg was added, and then the total volume was adjusted to 200 ml and shaken at 4 ° C. overnight. The supernatant was decanted and added to hinder buffer (1M ethanolamine, pH 8.0) and left at 4 ° C. for 16 hours.

Example 4: Purification of EPO Protein by Sepharose 4B Column with Monoclonal Antibody Attached

As a starting material, the EPO protein prepared by the gene recombination method was used. That is, the CHO cell line transformed with EPO gene (CJ Co., Ltd.) was cultured, and the culture solution was concentrated and used as a protein solution.

After filling 50 ml of Sepharose 4B with a monoclonal antibody attached to the fusion cell of Accession No. KCLRF-BP-00071 according to the method of Example 3, the buffer solution (50 mM sodium phosphate + 0.15 M NaCl) was added. , pH 7.0) and equilibrated with 0.3 L. 0.5 liter of the protein solution obtained above was passed through a column, washed with 300 ml of a buffer solution (50 mM sodium phosphate + 0.5 M NaCl, pH 7.0), and the protein was eluted with 32 ml of eluent (0.1 M citric acid). After adjusting the pH to 8.0.

Protein content in the solution was measured according to the Bradford method (Bradford, MM, Anal. Biochem. 72, pp248-254, 1976) using serum proteins as a standard, and the degree of purification was measured by Laemmli, UK, Nature 227, pp680 ~ 683, 1970) was confirmed by silver staining by electrophoresis on SDS polyacrylamide gel. The results are shown in FIG. Lane 2 in Figure 1 shows the results of electrophoresis of human EPO protein purified with monoclonal antibodies produced by fusion cells, Accession No. KCLRF-BP-00071.

Example 5: Purification of Modified Human EPO Protein by Sepharose 4B Column Attached with Monoclonal Antibody

As a starting material, a modified EPO protein (CJ Co., Ltd.) prepared by genetic recombination method was used. That is, the CHO cell line transformed with the modified human EPO gene was cultured, and the culture solution was concentrated and used as a protein solution.

Purification was carried out in the same manner as in Example 4, and the results are shown in FIG. Lane 3 in Figure 1 shows the results of electrophoresis of the modified human EPO protein purified by monoclonal antibody produced by fusion cells of Accession No. KCLRF-BP-00071.

Example 6: Western blot experiment using monoclonal antibodies

Western blot experiments were performed by the method of Tobin et al. (Towbin et al., PNAS 76, p4350, 1979). In other words, a purified solution containing human EPO protein and modified human EPO protein was electrophoresed on SDS-polyacrylamide gel (Novex) and then electrophoresed on nitrocellulose paper (25 mM Tris + 192 mM glycine + 20% methanol, pH 8.3). After nitrocellulose paper and gel were assembled, the mixture was added to a blot chamber, filled with the same buffer solution, and a current of 200 mA was passed for 6 hours. After removing the nitrocellulose paper, added to the buffer solution (0.1mM sodium phosphate + 0.15M NaCl + 3% BSA, pH 7.2) and left at room temperature for 1 hour, and then again by the fusion cell of accession number KCLRF-BP-00071 It was added to the same buffer containing the produced monoclonal antibody and left at room temperature for 2 hours.

After washing several times with the same buffer solution for 30 minutes, anti-mouse serum to which horse radish peroxidase was attached was added and left at room temperature for 30 minutes for color development. The results are shown in FIG. Lane 1 in Figure 2 shows the Western blot results of purified human EPO protein, lane 2 shows the Western blot results of purified modified human EPO protein.

Example 7: Isotype Determination of Monoclonal Antibodies

Immunopure Monoclonal Antibody Isotyping (Immunopure Monoclonal Antibody Isotyping) by Pierce (USA) to identify antibody isotypes of antibodies produced by fusion cells, Accession No. KCLRF-BP-00071 Experiment was carried out using Kit I). Experimental method is as follows.

50 μl of the coating antibody in the kit was added to each well and left at 4 ° C. overnight. The supernatant was removed and 125 μl of blocking solution (0.5% BSA / PBS) was added, left at 37 ° C. for 1 hour, and washed. 50 μl of cell culture was then added to each well and left for 1 hour at 37 ° C. before washing. Then 50 μl of subclass-specific anti-mouse immunoglobulin was added to each well, left at 37 ° C. for 1 hour, and washed four times. Thereafter, goat anti-rabbit IgG conjugated with horse radish peroxidase was added to each well and left at 37 ° C for 1 hour. After washing, 100 μl of ABTS substrate solution was added to each well and allowed to stand at room temperature for about 30 minutes to measure absorbance at 405 nm. As a result, the accession number of KCLRF-BP-00071 an arc, the monoclonal antibody produced by the fused cells was confirmed to be IgG type _3.

Fusion cells according to the present invention can produce a monoclonal antibody that recognizes both human EPO and modified human EPO,

According to the monoclonal antibody which recognizes both human EPO and modified human EPO according to the present invention, the separation, identification and purification of EPO and modified EPO, in particular, attached to a column to purify the EPO and modified EPO protein by chromatography It can be usefully used.

1 is a diagram showing the results of SDS-polyacrylamide gel electrophoresis of human EPO protein and modified human EPO protein purified by monoclonal antibody produced by fusion cell No. KCLRF-BP-00071, respectively. : Size marker, 2: purified human EPO protein, 3: purified modified human EPO protein.

FIG. 2 shows Western blot results using monoclonal antibodies produced by fusion cells of Accession No. KCLRF-BP-00071 for purified human EPO protein and modified human EPO protein, respectively. Human EPO protein, 2: purified modified human EPO protein.

Claims (3)

Fusion cell No. KCLRF-BP-00071. delete Monoclonal antibody produced by the fusion cell according to claim 1.