KR100701923B1 - - monoclonal antibody against b-lymphoma and hybridoma cell line producing the same - Google Patents

Abstract

A monoclonal antibody against B-lymphoma is provided to inhibit the activity of the B-lymphoma and the growth of the B-lymphoma cells by recognizing a protein HLA class II DR which is over-expressed in the B-lymphoma as an antigen, and being specifically bound thereto. The monoclonal antibody against B-lymphoma includes a heavy chain variable region having an amino acid sequence of sequence information 1, which is specifically bound to the B-lymphoma to inhibit the activity of the B-lymphoma and described as SEQ ID : NO. 1, and a light chain variable region having an amino acid sequence described as SEQ ID : NO. 2. The hybridoma cell line D56 producing the monoclonal antibody is deposited as a deposition number of KCLRF-BP-00128.

Description

MONOCLONAL ANTIBODY AGAINST B-LYMPHOMA AND HYBRIDOMA CELL LINE PRODUCING THE SAME}

1 shows ELISA results for Daudi cell line, a B-lymphoma cell line, Raji cell line, and SK-BR-3, a breast cancer cell line, for selected clone D56.

2 is a result confirming the binding to the B-lymphoma cell line through the cell analyzer of clone D56,

Figure 3 is the result of confirming the growth inhibition for the B-lymphoma cell line Raji and Daudi cell line,

4 shows western blotting results for purified antigen of clone D56

The present invention relates to an anti-B-lymphoma monoclonal antibody and a hybridoma cell line producing the same, more specifically, the protein HLA class II DR that is overexpressed on the surface of B-lymphoma is recognized as an antigen. , Monoclonal antibodies specifically binding thereto to inhibit the activity of B-lymphoma, its heavy chain variable region (V _H ) and its DNA, its light chain variable region (V _L ) and its DNA and its monoclonal antibody It relates to a hybridoma cell line producing.

Lymphoma is a primary malignant tumor of lymphoid tissue. Malignant lymphoma is a malignant tumor of lymphocytes in white blood cells. Although leukemia is a malignant tumor of lymphocytes called lymphocytic leukemia, it is different from malignant lymphoma in that the place where malignant cells increase is mainly blood and bone marrow (like plant that makes blood in bone).

Lymphocytes are a very important component of the human immune system, which is produced in the thymus, spleen and bone marrow (adult) and accounts for about 30% of all white blood cells present in the human (adult) circulation. Lymphocytes consist mainly of T cells and B cells, where T cells are responsible for cell mediated immunity and B cells are responsible for antibody production (bodily immunity).

B cells express different antibodies on their surfaces. Thus, one B cell expresses an antibody specific for one antigen and the other B cell expresses an antibody specific for another antigen. Thus, B cells are quite diverse and this diversity is important for the immune system. Each B cell in human can produce multiple antibody molecules (ie, about 10 ^{7-10 8} ). When foreign antigens are neutralized, such antibody production is almost usually stopped or substantially reduced. In some cases, however, certain B cell proliferation continues without diminishing, which can result in a cancer called B lymphoma.

Malignant lymphoma is the third most common tumor in childhood. Malignant lymphomas are roughly classified into Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL) due to differences in their biological properties. The two diseases occur in the same lymphoid tissue, but the clinical symptoms, course, pathophysiology and treatment methods are completely different. Especially in non-Hodgkin's lymphoma, there are many differences depending on the subtype.

Compared with the West, the overall incidence is similar, but Hodgkin's lymphoma, which is relatively effective in malignant lymphoma, accounts for 10-20%, which is lower than that of 35-45% in the West. many.

Treatment for malignant lymphoma has been used, such as radiotherapy, chemotherapy, antigen antibody therapy, their combination therapy, hematopoietic stem cell transplantation (including bone marrow transplantation), depending on the type and stage.

In case of Hodgkin's lymphoma, the cure rate is very high only by radiation therapy to lymphoid tissue or surrounding tissues that occur when the lesion is limited to the lymph gland tissue.However, non-Hodgkin's lymphoma is caused by systemic symptoms and various organs. Since the cure rate is not high by conventional treatment alone, the intensive combination chemothrapy, which is a combination of several recently developed drugs, is the basic treatment method.

If the distribution of lymphomas is local, radiotherapy may be efficient, but in the wider range of exposure, severe side effects such as bone marrow disorders may occur. Bone marrow disorders are caused by problems with blood production due to radiation therapy. Because of this, white blood cells are reduced, causing infection easily, or bleeding is caused by a decrease in platelets. Irradiation of the head and neck may cause stomatitis, salivary gland disorders, and taste disorders. Side effects may be more severe when combined with chemotherapy, and are reported to increase the likelihood of developing malignant tumors such as secondary leukemia.

Chemotherapy is administered by injecting an anticancer agent orally or intravenously or intramuscularly. Various anticancer drugs have been developed according to the type and stage of lymphoma. In the case of Hodgkin's lymphoma, the most popular method is nitrogen mustard, vincristine, procarbazine, or prednisone (MOPP) therapy, or ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) therapy. Examples include vincristine, cyclophosphamide, prednisone, methotrexate, and adriamycin. In addition, etoposide, bleomycin, L-asparaginase, and cytosine arabinoside are also used.

Whereas conventional radiotherapy is a topical therapy that is effective only in the place irradiated, chemotherapy is also called systemic therapy because chemotherapy can spread throughout the bloodstream and kill tumor cells. However, because chemotherapy is a systemic therapy, it is likely that all the organs of the body will have anti-cancer sequelae. Depending on the type of anticancer drug used, the number one side effect common to most anticancer drugs is myeloid toxicity (leukopenia, thrombocytopenia, anemia). Reduced white blood cells are more susceptible to infection and sometimes cause high fever. In such cases, antibiotics should be administered. When platelets decrease, bleeding is more likely to occur. Other major side effects include nausea, vomiting, loss of appetite, peripheral neuropathy (fouling limbs), constipation or diarrhea, liver and kidney disorders, hair loss, heart and lung disorders.

As mentioned above, traditional methods for treating B cell malignancies have been limited in their use due to toxic side effects.

As a new therapeutic method, monoclonal antibodies that recognize and respond to proteins expressed on the surface of B cells are continuously being studied.

The use of monoclonal antibodies is less toxic in normal tissues because these agents are selectively transported to and labeled or targeted to the tumor site. Therefore, it shows the highest effect in the tumor treatment of lymphatic system.

Proteins expressed on the surface of B cells in these B cell malignancies include CD19, CD20, CD21, CD22, and CD23 in B cells, and these are recognized as antigens and development of monoclonal antibodies against them has been studied.

Already, Biogen Idec of the United States has developed a monoclonal antibody targeting CD20, a cell surface protein that increases in tumor B cells, and sells it under the name Rituximab.

CD20 antigen is present in at least 80% of B cells of non-Hodgkin's lymphoma (NHL) patients. Rituximab is a monoclonal antibody that was first approved by the US FDA in 1997 as a cancer drug, and is a mouse antibody that targets the CD20 antigen, replacing the Fc region with human antibodies (light and heavy chain variable regions in mice). And gamma I heavy and kappa light chain insomnia regions in humans) to better respond to human complement and target cells and to reduce side effects of immunological responses. Initial studies reported a response rate of 50% and median time to progression of 10.2 months when used alone in 34 recurrent low-grade follicular lymphomas. In a large phase 3 study, 166 patients received a standard dose of Mabthera with a 48% overall response rate, including a 6% complete response, and a median time to progression of the responding patients was 13.2 months. Based on this study, Rituximab was approved by the FDA in 1997 for the treatment of relapsed or refractory low grade non-Hodgkin's lymphoma. Drugs that enhance the therapeutic effect by attaching radioisotopes to these monoclonal antibodies continue to be developed. In addition, CD19, CD20, CD21, CD22 and CD23, etc., and HLA class II DR that are overexpressed on the cell surface of B-lymphoma Antigen can also be a therapeutic target for malignant B cells, and research and development on this need to be conducted in various ways.

An object of the present invention is to recognize an anti-B lymphoma monoclonal antibody which inhibits the activity and growth of B-lymphoma by specifically binding to the antigen HLA class II DR overexpressed on the cell surface of B-lymphoma, 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 the amino acid sequence of SEQ ID NO: 1 specifically binding to B-lymphoma and inhibiting the activity of B-lymphoma and the amino acid sequence of SEQ ID NO: 2 Eggplant provides an anti-B lymphoma monoclonal antibody comprising a light chain variable region (V _L ).

In addition, the present invention provides a single anti-B lymphoma species comprising the 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 the light chain variable region (V _L ) Provides clone antibody DNA.

The present invention also provides a hybridoma cell line D56 (Accession No. KCLRF-BP-00128) for producing the monoclonal antibody.

The present invention also provides anti-B lymphoma monoclonal antibody D56 produced from the hybridoma cell line D56 (Accession No. KCLRF-BP-00128).

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

The present invention also provides a B-lymphoma therapeutic agent comprising the anti-B lymphoma monoclonal antibody.

Hereinafter, the present invention will be described in detail.

The present inventors inject the B-lymphoma cell line Daudi (human burkitt lymphoma, EBV-positive suspension cell) into the abdominal cavity of the mouse, and then immunized the mouse after immunizing the mouse by injecting the same cancer cells intraperitoneally three weeks later. Preparing a hybridoma cell line by mixing mouse spleen cells with myeloma cells (SP2 / 0) and incubating with pulley polyethylene glycol (molecular weight: 1,500; Roche); Hybridoma cell lines that secrete antibodies with high binding capacity are selected from the prepared hybridoma cell lines, and hybridoma cell lines that specifically react with B-lymphoma by limiting dilution to make the cell lines monoclonal. Screening; Mass production and purification of monoclonal antibodies from mice using the selected hybridoma cell line; Confirming reactivity in the B-lymphoma cell line through a flow cytometer; Identifying growth inhibition of Daudi and Raji, B-lymphoma cell lines; Determining the antigen of the growth inhibitory B-lymphoma monoclonal antibody; 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: B- lymphoma  To produce monoclonal antibodies Hybridoma  build

The Daudi cell line, a B-lymphoma 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 Daudi cell line was boosted into the abdominal cavity of mice. Four days before the cell fusion experiment, Daudi cell lines were 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: to produce monoclonal antibodies Hybridoma  Selection of cells

Among the hybridoma cell groups prepared in Example 1, selection of hybridoma cell lines that specifically respond to B-lymphoma was performed by measuring the antibody binding to B-lymphoma using the ELISA method as follows. First, dilute the Daudi membrane extract with 10 μg / ml phosphate buffered saline (PBS), 100 μl of this diluted Daudi membrane extract was dispensed into 96-well plates (Nunc Immuno Module, Maxisorp, Denmark) and incubated overnight at 4 ° C. It was. 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) One clone was selected and named D56. The hybridoma cell line D56, which produces the monoclonal antibody D56, was deposited to the Korean Cell Line Research Foundation (KCLRF) on January 23, 2006. (Identification reference: Daudi-56-2B7). (Accession No. KCLRF-BP-00128)

1 shows ELISA results for the selected clone D56, Daudi cell line, a B-lymphoma cell line, Raji (human burkitt lymphoma, EBNA positive suspension B cell) cell line, and SK-BR-3, a breast cancer cell line.

As shown in FIG. 1, it can be seen that B-lymphoma cell lines, Daudi cell line and Raji cell line, show strong binding ability.

Example  3: Production and Purification of Monoclonal Antibodies

In order to produce monoclonal antibody from the hybridoma cell line continuously secreting the antibody obtained in Example 2, 0.5 ml of 2,6,10,14-tetramesylpentadedecane (pristane, Sigma) 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: using a cytometer B- lymphoma The binding for

In order to confirm the binding to B-lymphoma against monoclonal antibody D56 confirmed to be reactive against B-lymphoma in Example 2, each of the B-lymphoma cell lines Daudi cell line and Raji cell line were treated with 1% bovine serum albumin (BSA). Washed with PBS. 10ug of monoclonal antibody was reacted with B-lymphoma 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 2 shows the results confirming the binding to the B-lymphoma cell line through the cell analyzer of clone D56.

As shown in FIG. 2, the monoclonal antibody D56 was confirmed to have excellent reactivity with Daudi and Raji cells.

Example 5: Growth Inhibition Experiment on B-lymphoma Cell Line

To confirm the growth inhibition experiment of B-lymphoma cells against monoclonal antibody D56 that binds to B-lymphoma cell line in Example 4, Raji and Daudi cells were placed in 12-well microplates (Nunc) to 2 × 10 ⁵ cell counts. D56 monoclonal antibody was then treated.

Figure 3 is the result of confirming the growth inhibitory ability for B-lymphoma cell lines Raji and Daudi cell lines.

As shown in FIG. 3, the change in cell number was measured daily, and it was confirmed that monoclonal antibody D56 had superior growth inhibitory ability against Raji and Daudi cell lines compared to TT-2, which is an anti-tetanus monoclonal antibody.

Example 6: Monoclonal Antibodies D56 Antigen Identification

The monoclonal antibody D56 purified in Example 3 was reacted with CNBr activated sepharose (Amersham Pharmacia) to prepare affinity resin, and then the antigen-binding antigen was isolated from Daudi extract. The binding to the antigen was confirmed (FIG. 4), and the antigen separated by Maldi-TOF was identified. It was identified as HLA class II DR antigen.

4 shows western blotting results for the purified antigen of monoclonal antibody D56.

Example  7: B- lymphoma  For cell lines Growth inhibition  Identified Monoclonal Antibodies D56 DNA base sequence determination

In order to determine DNA sequencing for monoclonal antibody D56, which has been shown to inhibit the growth of B-lymphoma cell line in Example 5, hybridomas were cultured in DMEM medium at about 1 × ^{10 8} , and the results were described using Trizol (Life Technologies). RNA was isolated accordingly. 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 cultured 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 B-lymphoma monoclonal antibody of the present invention recognizes B-lymphoma that is overexpressed in various types of tumors or cancers as an antigen, specifically binds to inhibit the activity of B-lymphoma, thereby growing 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 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 B-lymphoma to inhibit the activity of B-lymphoma Anti-B lymphoma monoclonal antibody. Anti-B lymphoma 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-B lymphoma monoclonal antibody D56 according to claim 1, wherein said monoclonal antibody is produced from hybridoma cell line D56 (Accession No. KCLRF-BP-00128). The hybridoma cell line D56 producing the monoclonal antibody of claim 1 (Accession No. KCLRF-BP-00128). The recombinant antibody expressed in nucleotide sequence information of SEQ ID NO: 3 and SEQ ID NO: 4 of claim 2. A therapeutic agent for malignant B lymphoma, comprising the anti-B lymphoma monoclonal antibody of claim 1.

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