KR100680141B1 - - monoclonal antibody against epidermal growth factor receptor and hybridoma cell line producing the same - Google Patents

Abstract

The present invention relates to an anti-epidermal growth factor receptor (EGF receptor or EGFR) monoclonal antibody and a hybridoma cell line producing the same. More specifically, the present invention relates to an EGF receptor specifically binding to an EGF receptor. The present invention relates to a monoclonal antibody that inhibits activity, a heavy chain variable region thereof (V _H ) and a DNA encoding the same, a light chain variable region (V _L ), a DNA encoding the same, and a hybridoma cell line producing the monoclonal antibody. The anti-EGF receptor monoclonal antibody of the present invention has the effect of inhibiting the growth of tumor cells by inhibiting the activity of the EGF receptor by specifically binding to EGF receptors that are overexpressed in various types of tumors or cancers as antigens. .

Epidermal growth factor receptor, EGF receptor, EGFR, monoclonal antibody, hybridoma cell line

Description

MONOCLONAL ANTIBODY AGAINST EPIDERMAL GROWTH FACTOR RECEPTOR AND HYBRIDOMA CELL LINE PRODUCING THE SAME}

1 shows ELISA results for the A431 cell line, which is an EGF receptor and an EGF receptor overexpressing cell line, for selected clones.

2 is an immunohistostaining result of clone A431-1 and clone A431-96,

3 is a result of confirming the binding of EGF receptor through the cell analyzer of clone A431-1 and clone A431-96,

4 is a western blotting result confirming the phosphorylation inhibitory activity of the EGF receptor, lane 1 is showing the molecular weight, lane 2 is MDA-MB-231 cell lysate without any treatment, lane 3 is EGF-only cell lysate, lane 4 Is a cell lysate treated with anti-tetanus antibody, EGF-treated, lane 5 treated with clone A431-1, EGF-treated cell lysate, lane 6 treated with clone A431-96, and treated with EGF.

Figure 5 confirms the growth inhibition of the lung cancer cell line NCI-H727 cell line.

The present invention relates to an anti-epidermal growth factor receptor (EGF receptor or EGFR) monoclonal antibody and a hybridoma cell line producing the same. More specifically, the present invention relates to an EGF receptor specifically binding to an EGF receptor. The present invention relates to a monoclonal antibody that inhibits activity, a heavy chain variable region thereof (V _H ) and a DNA encoding the same, a light chain variable region (V _L ), a DNA encoding the same, and a hybridoma cell line producing the monoclonal antibody.

Epidermal Growth Factor Receptor (EGFR, hereinafter referred to as 'EGF receptor') has been found to be overexpressed in various types of tumors. Overexpression of the EGF receptor has been observed, for example, in certain cancers, lung cancer, breast cancer, colon cancer, gastric cancer, brain cancer, bladder cancer, head and neck cancer, ovarian cancer and prostate cancer. Epidermal Growth Factor (EGF) has been shown to bind to EGF receptors and induce cell proliferation and tumor growth. Therefore, inhibiting the binding of EGF to tumor cells overexpressing the EGF receptor using an antibody against the EGF receptor can inhibit cancer cell growth and treat cancer. Various mouse and rat monoclonal antibodies against the EGF receptor have been developed and tested for their ability to inhibit tumor cell growth in vitro and in vivo.

C225 antibody (product name: Erbitux, ImClone, USA), which is used in the treatment of metastatic colorectal cancer in the clinical field, is a chimeric antibody that combines the variable region of mouse with the IgG1 constant region of human antibody. And inhibits phosphorylation of EGF receptors by EGF and inhibits human tumor formation in nude mice. The antibody has also been shown to synergize with certain chemotherapeutic agents to eradicate human tumors in xenograft mouse models.

An object of the present invention is an anti-EGF receptor monoclonal antibody which inhibits the growth of tumor cells by inhibiting the activity of the EGF receptor by specifically binding to the antigen, specifically binding to EGF receptor overexpressed in various types of tumors or cancers and the same It is to provide a hybridoma cell line to produce.

In order to achieve the above object, the present invention provides a heavy chain variable region (V _H ) having an amino acid sequence of SEQ ID NO: 1 specifically binding to an EGF receptor to inhibit the activity of the EGF receptor, and a light chain having an amino acid sequence of SEQ ID NO: 2 It provides an anti-EGF receptor monoclonal antibody comprising a variable region (V _L ).

In addition, the present invention provides a single anti-EGF receptor comprising a nucleotide sequence of SEQ ID NO: 3 encoding the monoclonal antibody heavy chain variable region (V _H ) and the nucleotide sequence of SEQ ID NO: 4 encoding a light chain variable region (V _L ) Provides clone antibody DNA.

The present invention also provides a hybridoma cell line A431-96 (Accession No. KCLRF-BP-00114) for producing the monoclonal antibody.

The present invention also provides an anti-EGF receptor monoclonal antibody A431-96 produced from the hybridoma cell line A431-96 (Accession No. KCLRF-BP-00114).

The present invention also provides a recombinant antibody expressed in the nucleotide sequence information of SEQ ID NO: 3 or SEQ ID NO: 4.

The present invention also provides a cancer therapeutic agent using the anti-EGF receptor monoclonal antibody.

Hereinafter, the present invention will be described in detail.

The present inventors inject the breast cancer cell line MDA-MB-231 into the abdominal cavity of the mouse, and after 3 weeks, the same cancer cells were boosted intraperitoneally, and the skin cancer cell line A431, intraperitoneally, was boosted before the cell fusion. After immunizing the splenocytes of the immunized mouse mixed with myeloma cells (SP2 / 0) and the polyethylene glycol (PEG, molecular weight 1,500; Roche) and reacted and cultured to prepare a hybridoma cell line ; Among the hybridoma cell lines prepared, hybridoma cell lines that secrete antibodies with high binding capacity are selected, and the limiting dilution is performed so that the cell lines become monoclonal cells. Screening; Mass production and purification of monoclonal antibodies from mice using the selected hybridoma cell lines; Confirming reactivity in tissues in which the EGF receptor is overexpressed by immunohistostaining; Confirming reactivity in cancer cell lines in which EGF receptors are overexpressed through a flow cytometer; Confirming the phosphorylation inhibitory activity of the EGF receptor; Identifying growth inhibition in NCI-H727 cell line, which is a lung cancer cell line in which EGF receptor is expressed; The present invention has been completed through the step of identifying the DNA sequence for the monoclonal antibody having growth inhibition.

Hereinafter, the present invention will be described in detail through examples. This embodiment is only a preferred embodiment, the present invention is not limited thereto.

Example 1 Construction of Hybridomas Producing Anti-EGF Receptor Monoclonal Antibodies

MDA-MB-231 cell line, a breast cancer cell line, was well mixed with phosphate buffer and injected into the abdominal cavity of 4 week old Balb / c mice. Three weeks later, the MDA-MB-231 cell line was boosted into the abdominal cavity of mice. Four days before the cell fusion experiment, the A431 cell line, a skin cancer cell line, was mixed well with phosphate buffer and injected into the abdominal cavity of mice. Spleens of immunized mice were isolated and splenocytes isolated. The separated splenocytes and myeloma cells (SP2 / 0) were washed twice with DMEM medium, and the splenocytes and myeloma cells were counted and mixed so that the splenocytes and myeloma cells were 5: 1. Then wash once. Add 1 ml of polyethylene glycol, mix, add DMEM medium, centrifuge and suspend the precipitated cells in HAT medium, and spread 0.2 ml each on a 96-well micro titer plate. C was incubated in a carbon dioxide incubator.

Example 2: Selection of Hybridoma Cells Producing Monoclonal Antibodies

Among the hybridoma cell groups prepared in Example 1, a hybridoma cell line that specifically reacts to the EGF receptor (Sigma, USA) is selected by using an ELISA method to bind an antibody to the EGF receptor (Sigma). It was made by measuring as follows. First, the EGF receptor was diluted with 2 μg / ml phosphate buffered saline (PBS), and 100 μl of this diluted EGF receptor was aliquoted into 96 well plates (Nunc Immuno Module, Maxisorp, Denmark) and incubated at 4 ° C. overnight. Plates were added with 300 μl of 1% bovine serum albumin (BSA) PBS and incubated for 1 hour at room temperature. After washing, 100 μl of hybridoma culture solution was added thereto, and the cells were incubated at room temperature for 2 hours. After a second wash with PBST, diluted goat anti-mouse IgG (Fab specific) -peroxidase conjugate (Sigma, USA) in 1% BSA-PBS was added and incubated for 1 hour at room temperature. Wash once more with PBST, then add 100 μl of 2,2′-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid (ABTS) solution (KPL, USA) and absorbance at 405 nm (OD) 9 clones were selected by measuring.

1 shows ELISA results for A431 cell line, which is an EGF receptor and an EGF receptor overexpressing cell line, for selected clones.

Among them, monoclonal antibodies A431-1 and A431-96, which are responsive to the skin cancer cell line A431 extract and the EGF receptor, were selected. Among them, the hybridoma cell line A431-96 producing monoclonal antibody A431-96 was selected in 2005. On March 25, it was deposited with the Korean Cell Line Research Foundation (KCLRF) (Accession No. KCLRF-BP-00114).

Example 3 Production and Purification of Monoclonal Antibodies

0.5 ml of 2,6,10,14-tetramesylpentadecane (pristane, Sigma) in mice (Balb / c) to produce monoclonal antibodies from the continually secreting hybridoma cell line obtained in Example 2 G) was injected intraperitoneally. After one week, each hybridoma cell line was injected 5 x 10 ⁶ per mouse, and ascites fluid was collected from mice inflated intraperitoneally. Freeze this ascites solution at -20 ℃ and remove the part that is not frozen After dissolving the ascites solution, precipitate with ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and then centrifuge to dissolve the precipitate with phosphate buffer. Then, the mixture was mixed 1: 1 with antibody purification binding buffer (Pierce, USA) and purified using a column filled with protein A-agarose gel.

Example 4: Confirmation of EGF receptor binding using immunohistostaining of monoclonal antibodies

In order to confirm the binding of EGF receptors using the immunohistostaining method of the purified monoclonal antibodies in Example 3, placental tissue slide (Spring Bioscience, USA), which is an overexpressed EGF receptor, was first subjected to xylene for three times for 10 minutes. Paraffin was removed by soaking. Paraffin-free slides were soaked in ethanol and washed for 5 minutes in tap water. The slides were then placed in 10 mM citrate pH6.0 buffer, discontinued and immersed in 3% hydrogen peroxide to inactivate peroxidase in the tissues. Monoclonal antibody 1ug / mL was mixed in phosphate buffer solution, soaked in a slide and incubated for 1 hour at room temperature. After incubation, the cells were washed three times with phosphate buffer, and then goat anti-mouse IgG (Fab specific) -peroxidase conjugate (NICHIREI, Japan) was added and incubated at room temperature for 30 minutes. StableDAB peroxidase substrate (KPL Inc.) was added to check the degree of staining under a microscope and washed with tap water for 5 minutes. It was dyed with Contrast BLUE solution (KPL) for 1 minute and then washed with tap water. The slides were soaked in ethanol and then soaked three times in xylene for 2 minutes. It was then mounted in Permount (Fisher, USA) and stored.

2 shows immunohistostaining results of clone A431-1 and clone A431-96.

As shown in FIG. 2, the experimental results confirmed that the clone A431-1 and clone A431-96 were stained on the placental tissue slide.

Example 5 Confirmation of Binding to EGF Receptor in Cancer Cell Lines Overexpressing EGF Receptor Using a Cell Analyzer

In order to confirm the binding to the cancer cell line overexpressing the EGF receptor for the monoclonal antibody confirmed the reactivity to the EGF receptor in Example 4, washed the A431 cell line, a skin cancer cell line with 1% bovine serum albumin (BSA) PBS gave. 10ug of monoclonal antibody was reacted with A431 cell line in ice and washed twice with 1% BSA-PBS. Goat anti-mouse (Fab specific) FITC conjugate was added and reacted for 40 minutes on ice and washed twice with 1% BSA-PBS. It was suspended in 1 mL of 1% BSA-PBS and analyzed by flow cytometry.

Figure 3 shows the results of confirming the binding of EGF receptor through the cell analyzer of clone A431-1 and clone A431-96.

As shown in FIG. 3, experiments showed that clone A431-1 and clone A431-96 responded to A431 cells but did not react to hTT-2, an anti-tetanus monoclonal antibody.

Example 6 Inhibition of EGF Receptor Phosphorylation in Breast Cancer Cells

In order to confirm the EGF receptor phosphorylation inhibitory ability of the monoclonal antibody binding to the EGF receptor in Example 5, MDA-MB-231 cell line, which is a breast cancer cell line, was laid in a 24-well microplate (Nunc) to have a cell number of 1 × 10 ⁵ . After 2 days, the monoclonal antibody and EGF were treated, and after 30 minutes, the MDA-MB-231 cell lysate was separated from the SDS-PAGE protein band and transferred to the nitrocellulose membrane by electric method. Anti-phosphotyrosine specific peroxidase conjugates that block membranes with 5% bovine serum albumin solution for 30 minutes and specifically respond to phosphorylated forms of EGF receptors to reduce nonspecific responses of proteins transferred to membranes (Zymed, USA) Was reacted at 4 ° C. overnight. After washing with phosphate buffer containing 0.05% Tween, 0.018% (v / v) 4-chloro-1-naphthol and 0.045% hydrogen peroxide were colored using phosphate buffer and a substrate dissolved in methanol.

4 is a western blotting result confirming the phosphorylation inhibitory activity of the EGF receptor, lane 1 is showing the molecular weight, lane 2 is MDA-MB-231 cell lysate without any treatment, lane 3 is EGF-only cell lysate, lane 4 Is a cell lysate treated with anti-tetanus antibody, EGF-treated, lane 5 treated with clone A431-1, EGF-treated cell lysate, lane 6 treated with clone A431-96, and treated with EGF.

As shown in FIG. 4, the experimental results confirmed that clone A431-1 and clone A431-96 inhibit phosphorylation of the EGF receptor.

Example 7: Growth inhibition experiment of EGF receptor expressing lung cancer cells

In Example 6, 0.5% agar was hardened in a 24-well microplate (Nunc) to confirm growth inhibition of skin cancer cells against monoclonal antibody having phosphorylation inhibition of the EGF receptor, and the lung cancer cell line NCI-H727 cells and Monoclonal antibodies were mixed and mixed in a medium containing 0.3% agar. After 3 weeks, the living cells were stained using nitro blue tetrazolium (Sigma).

Figure 5 confirms the growth inhibition of the lung cancer cell line NCI-H727 cell line.

As shown in FIG. 5, the experimental results confirmed that clone A431-96 had superior growth inhibitory ability against NCI-H727 cell line as compared to hTT-2, which is an anti-tetanus monoclonal antibody.

Example 8: Determination of DNA base sequence of monoclonal antibody with growth inhibition ability against lung cancer cell line

In order to determine DNA sequencing for monoclonal antibodies in which growth inhibition of lung cancer cell lines was confirmed in Example 7, hybridomas were cultured in DMEM medium at about 1 × ^{10 8} , and RNA was prepared according to instructions using Trizol (Life Technologies). Separated. Cloning of antibody genes, such as cDNA synthesis and amplification of V _H and V _L DNA of antibodies, was carried out according to the methods presented using the Mouse ScFv and Expression Module (Amersham Pharmacia). After inserting into the T vector (Promega, USA), the nucleotide sequence was confirmed. The detailed process is as follows.

1. Isolation of RNA

1 ml of trizol per 5-10 × 10 ⁶ hybridoma cells was added to the cells, followed by pipetting, and the cells were waited at room temperature (15 ° C.-30 ° C.) for 5 minutes. Next, 200 μl of chloroform per 1 ml of Trizol was mixed well, the lid was closed and shaken vigorously for 15 seconds by hand, and then waited at room temperature for 3 minutes. Thereafter, the mixture was centrifuged at 15,000 rpm for 15 minutes at 2 to 8 ° C, and only the supernatant was transferred to a new tube. 500 μl of isopropyl alcohol was added per 1 ml of Trizol, mixed well, and then waited at room temperature for 10 minutes. Thereafter, the mixture was centrifuged at 15,000 rpm for 10 minutes at 2 to 8 ° C, and the supernatant was removed. 1 ml of 75% ethanol was added per 1 ml of trizol, and centrifuged at 15,000 rpm for 5 minutes at 2 to 8 ° C. 150 μl of distilled water was dissolved in the RNA, and then the absorbance was measured and measured at 260 nm. It was then stored at -20 ° C. The amount of RNA isolated was 1.0-2.5 μg / μl.

2. Synthesis and Amplification of cDNA

CDNA was synthesized from the RNA obtained above. First, mRNA was reacted at 65 ° C. for 10 minutes and immediately cooled on ice. 10 μl of mRNA, 10 μl of distilled water, 11 μl of a first-strand mix, and 1 μl of DTT were added and reacted at 37 ° C. for 1 hour. Herein, the first strand mixture is included in the Mouse ScFv and Expression Module (Amersham Pharmacia), a mouse reverse transcriptase, random hexadeoxyribonucleotides (pd (N) ₆ ) , BSA, dATP, dCTP, dGTP, dTTP without RNase / DNase.

Next, PCR was performed using the obtained cDNA. Two PCR tubes were prepared, and one tube was filled with a PCR kit, 33 µl of cDNA, 2 µl of a light primer mix and 15 µl of distilled water, and the other tube contained a PCR kit, 33 µl of a cDNA and a heavy chain primer. 2 μl of the mixture (heavy primer mix) and 15 μl of distilled water were added thereto, and the reaction was performed by a PCR machine. The PCR reaction was carried out by reacting for 5 minutes at 95 ° C., then 40 times with 1 minute at 95 ° C., 2 minutes at 56 ° C., 2 minutes at 72 ° C., and finally at 72 ° C. for 15 minutes. The light chain primer mixture refers to a mixture of primers corresponding to ten variable regions of the light chain, and includes a 5 'primer and a 3' primer. The heavy chain primer mixture is a mixture of primers corresponding to the variable region of the heavy chain, each present as a 5 'primer and a 3' primer, and was mixed and used together during the reaction. The mixtures themselves are provided to the Mouse ScFv and Expression Module (Amersham Pharmacia).

The PCR product DNA thus obtained was confirmed by electrophoresis on 1.2% agarose gel and separated and purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA).

3. Preparation of Recombinant Vectors

The recombinant VH and VL DNA obtained above and the pGEM-T vector were prepared using a T4 DNA ligase (Promega). The recombinant vector thus prepared was transformed into E. coli cells and plated on LB plates containing X-Gal and IPTG. After incubation at 37 ℃ overnight, white colonies were selected and incubated in LB medium, and recombinant plasmids were isolated using a Qiagen plasmid mini kit (Qiagen, Valencia, CA, USA). The recombinant plasmid DNA thus obtained was used for sequencing.

As described above, the anti-EGF receptor monoclonal antibody of the present invention recognizes and specifically binds an EGF receptor that is overexpressed in various tumors or cancers as an antigen, thereby specifically inhibiting the activity of the EGF receptor to grow tumor cells. It has the effect of suppressing. Therefore, the present invention can be usefully used for the treatment of various tumors and cancers.

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Claims (6)

A term comprising 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 specifically binding to the EGF receptor to inhibit the activity of the EGF receptor -EGF receptor monoclonal antibody. An anti-EGF receptor monoclonal antibody DNA comprising the nucleotide sequence of SEQ ID NO: 3 encoding the heavy chain variable region (V _H ) of claim 1 and the nucleotide sequence of SEQ ID NO: 4 encoding the light chain variable region (V _L ) of claim 1 . The anti-EGF receptor monoclonal antibody A431-96 according to claim 1, wherein the monoclonal antibody is produced from hybridoma cell line A431-96 (Accession No. KCLRF-BP-00114). The hybridoma cell line A431-96 (Accession No. KCLRF-BP-00114) which produces the monoclonal antibody of Claim 1. The recombinant antibody expressed in nucleotide sequence information of SEQ ID NO: 3 or SEQ ID NO: 4 of claim 2. A cancer therapeutic agent using the anti-EGF receptor monoclonal antibody of claim 1.

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