JPH07163392A - Human monoclonal antibody and hybridoma proudcing the same antibody - Google Patents

Abstract

PURPOSE:To obtain a new human monoclonal antibody specifically recognizing a surface antigen having a specific molecular weight of a cancer cell, reacting with a various kinds of cancer cells, useful for diagnosing and treating various kinds of cancers, having no antigenicity and little clinical adverse effect. CONSTITUTION:Cultured human stomach cancer cell line KATO-III is subcutaneously administered to a healthy person once in a month. Four days after the final administration, a peripheral blood is collected, lymphocyte separated from the peripheral blood is subjected to cell fusion with human B lymphoblastma cell line HO-323 by using polyethylene glycol and then selectively cultured in HAT medium to give a hybridoma. Then the hybridoma is screened by using KATO-III, a positive clone is selected and cloned by limiting dilution and the monocloned hybridoma is cultured to give the objective human monoclonal antibody specifically recognizing a surface antigen of cancer cell 56Kd, reacting with a various kinds of cancer cells, useful for diagnosing and treating various kinds of cancers.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human type monoclonal antibody which specifically binds to cancer cells, and a hybridoma which produces the antibody.

[0002]

2. Description of the Related Art A number of specific monoclonal antibodies against various human cancers have been produced by the method of Kohler-Milstein cell fusion using lymphocytes and antibody-producing partner cells. These monoclonal antibodies are currently used for cancer diagnosis and the like. However, most of these antibodies are of mouse origin and have antigenicity when administered to humans, so their use in clinical aspects was limited. Therefore, it has been desired to produce many useful human-human monoclonal antibodies having no antigenicity, but almost no such monoclonal antibody has been produced so far. This is due to the difficulty in obtaining good lymphocytes. The main starting material for human monoclonal antibodies is cancer patient lymphocytes,
This is because the probability of obtaining lymphocytes that produce specific antibodies against cancer antigens is very low.

The inventors of the present invention have conducted extensive research and obtained the following findings. (Koyama, K, et al., Jpn.J.Canc
er. Res., 81 967 (1990)). That is, healthy people were immunized with human gastric cancer cell lines MKN-45 and MKN-28, and peripheral blood lymphocytes and lymphoblastoma-like cell line HO-323 (Ohas
hi H, et al., Cell Biology International Reports, 10
77 (1986)) and many hybridomas were created. FMK-H3 (IgM-type) and EMK-F7 among them
The two (IgG-type) hybridomas are MKN-45 and MKN-28.
Produced a monoclonal antibody that strongly reacts with. However, the monoclonal antibodies obtained from FMK-H3 and EMK-F7 have a problem in that they are difficult to use for general cancer diagnosis because the number of reactive cancer cells is limited.

[0004]

The object of the present invention is to react with a greater variety of cancer cells than the conventionally known human monoclonal antibodies, and thus is useful for the diagnosis and treatment of multiple types of cancer. It is intended to provide an antibody and a hybridoma producing the monoclonal.

[0005]

Means for Solving the Problems That is, the present invention is a humanized monoclonal antibody that specifically recognizes a 56 Kd surface antigen of cancer cells. In addition, hybridoma GMK-A, GM
The above-mentioned monoclonal antibody produced by K-C3 or KMK-41. Furthermore, the present invention is a hybridoma that produces a humanized monoclonal antibody that specifically recognizes a 56 Kd surface antigen of cancer cells. The hybridoma includes human-human hybridoma, and further includes hybridoma GMK-A, GMK-C3 or KMK-41.

The present invention will be described in detail below. The present inventors have found that KATO-III (a cell line established from a signet ring cell in which human adenocarcinoma of the stomach is slightly differentiated,
Sekigichi M, et al., Jpn.J.Exp.Med., 48 61 (1978)))
And Hanganutziu-Deicher as indicators of cell surface glycolipids
(HD) Antigen (neuraminyl lactosylceramide, NeuGc-
GM3) was mixed, and the mixture was administered to a healthy person. Then, the screening of the monoclonal antibody produced by the hybridoma of the peripheral blood lymphocytes of the healthy person and the lymphoblastoma-like cell line HO323 is carried out by using KATO-III as the target cell to produce three types of human monoclonal antibodies. Hybridomas GMK-A, GMK-C3, and KMK-41 were obtained.

Next, the reactivity of the monoclonal antibody produced by the hybridoma to cancer cells was assayed by ELISA, and it was confirmed that these monoclonal antibodies bind to many types of cancer cells. further,
It was confirmed by immunoblotting that these monoclonal antibodies specifically react with the surface antigen of 56 Kd of cancer cells. Furthermore, it was confirmed that these monoclonal antibodies lose their reactivity to the 56Kd surface antigen of cancer cells when the sugar chain is modified to clarify whether or not the sugar chain is involved in the antigenicity and the sugar chain is oxidized. It was confirmed that the 56 Kd surface antigen was a glycoprotein.

Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited by this embodiment.

[0009]

【Example】

A. Method 1. Preparation of parent cells Cultured human gastric cancer cell line KA used to administer to healthy subjects
TO-III was cultured in RPMI1640 culture medium containing 2% serum of the same healthy person, and collected by scraper and centrifugation. The antigen is
The purified NeuGc-GM3 suspended in PBS and the cultured cells KATO-III were mixed and adjusted just before the administration. 1 ml PBS, 107 cells KATO-II
A mixed solution of I and 1000 μg NeuGc-GM3 was subcutaneously administered once a month. Lymphocytes are 107 cells KATO-III, 1000 μg Neu
Each time Gc-GM3 was administered, peripheral blood of a healthy individual collected 4 days after the administration was obtained by Ficoll-Paque density gradient centrifugation. Partner cells are HO-323 (6-thioguanine-resistant human B lymphoblastoma, Ohashi H, et al., Cell Biology Internation
al Reports, 10 77 (1986)) cell line was used.

2. Cell fusion, ELISA Cell fusion is polyethylene glycol (MW4000)
Was performed using. Hybridomas were selected by HAT medium (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM).
ERDF / 15% FCS medium containing M thymidine), and antibody screening was performed using E targeting KATO-III.
It was performed by LISA. Hybridomas were diluted to the limit, and immunoglobulins were detected using the Houghton ELISA method (Houghton ELISA method).
on, AN, et al., J. Exp. Med., 158, 53 (1983)). The reactivity of the monoclonal antibody against the cancer cell line was examined against the following cell lines: 10 gastric cancer cell lines (MKN-1, MKN-7, MKN-28, MKN-45, MKN-7.
4, TMK-1, NUGC-2, NUGC-3, NUGC-4, KATO-III), lung cancer 2
Species (PC-8, QG-56), Breast cancer 1 species (MCF-7), Bladder cancer 1
Species (KU-1), one epithelial cell carcinoma (A-431), and one fibroblast (FLOW-2000) were examined. These cell lines were cultured to a confluent state in 10% FCS-RDF culture medium and fixed with 0.06% glutaraldehyde in PBS. ELISA for monoclonal antibody reactivity
Considered in. Hybridomas that produce antibodies include insulin (final 5 μg / ml), transferrin (final
Serum-free EDRF medium containing 20 µg / ml), ethanolamine (final 20 µM), sodium selenium (final 2.5 nM), and egg yolk lipoprotein (final 15 µg / ml) was added, and the cells were further cultured for 4 days.

3. Immunoblotting Immunoblotting and periodate oxidation for antigen analysis were performed as follows. The above cell line 3 × 10 6 cells were sonicated and then subjected to SDS-PAGE. Gel transfer was performed on a nitrocellulose membrane. When it is necessary to denature the sugar chain of the antigen, the transferred filter is incubated with 50 mM sodium metaperiodate 50 mM sodium acetate buffer (pH 4.5) for 1 hour,
Further, it was incubated with 50 mM sodium borate PBS for 30 minutes. The reactivity of the culture supernatant was measured using the activity of 10% FCS-ERDF medium as a control. The culture supernatant of the hybridoma was blocked with 1% FCS-PBS and then reacted with a nitrocellulose membrane for 1 hour. After washing, it was reacted with an anti-human IgG antibody having peroxidase added as a secondary antibody for 1 hour. After washing, the reaction mixture was developed by adding DAB solution.

B. Result 1. Acquisition of hybridoma An abscess with neutrophil and lymphocyte infiltration was formed around the site where KATO-III 107 cell, 100 μg NeuGc-GM3 was subcutaneously administered, and tumor necrosis was observed in these abscesses. The abscess was absorbed within 3 weeks. 3 by cell fusion experiment
From the 556 hybridomas obtained from 7 collections 2 months after the first administration, 3 IgM hybridomas, G
Established MK-A, GMK-C3 and KMK-41. These hybridomas have been deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST. The deposit number of each hybridoma is as follows.

Hybridoma GMK-A FERM P-13669 Hybridoma GMK-C3 FERM P-13670 Hybridoma KMK-41 FERM P-13671 2. Monoclonal Antibody Reactivity The reactivity of monoclonal antibodies produced by these hybridomas against various cell lines confirmed by ELISA is shown in Table 1. MAb / GMK-A reacted with all cell lines used. MAb / GMK-C3 is also a gastric cancer cell line NUGC-2
And lung cancer cell lines other than QG-56. MAb / KMK-41 reacted strongly with all cell lines. Similar assays were performed on the previously established monoclonal antibodies produced by EMK-H3 and FMK-F7. FMK-H3 specific for gastric cancer cell line MKN-45, MKN-28
(IgM) is breast cancer cell line MPC-7, lung cancer cell line PC-8, Q
G-56, epithelial cancer cell line A-431, bladder cancer cell line K
It also responded to U-1, but not to other cells. . E
MK-F7 (IgM) responded only to the gastric cancer cell line MKN-45, especially MKN-28, but not to other cell lines. Table 1 Reactivity of human monoclonal antibody to human cancer cell line by ELISA Human antibody cell line from hybridoma FMK-H3 EMK-F7 GMK-A GMK-C3 KMK-41 ─────────────── ───────────────────── Stomach cancer MKN-1--++ ++ +++ MKN-7--++ ++ +++ MKN-28 +++ +++ ++ ++ +++ MKN-45 ++ +++ ++ ++ +++ MKN-74 +-++ ++ +++ TMK-1--++ ++ + ++ NUGC-2--++ + +++ NUGC-3--++ +++ +++ NUGC-4--++ ++ +++ KATO-III--++ ++ +++ Breast cancer MCF-7 ++ +++ +++ +++ Lung cancer PC-8 +++-+++ ++ +++ QG-56 ++-++ + +++ Epidermal cancer A-431 + ++-+++ ++ +++ Bladder cancer KU-1 +++-+++ ++ +++ Fibroblast Flow 2000--+++ ++ +++ ──────── ─────────────────────────── In Table 1, the reactivity intensity of the monoclonal antibody is as follows. Displayed as; absorbance at 405nm is 0.05 or less (-); 0.05-0.1 (+); 0.1-0.2 (++); 0.2 or higher (++
+). The reaction was performed using 2,2'-azino-bis (3-ethylbenzoth as a substrate.
iazoline-6-sufonic acid) diammonium salt was added and started. After 15 minutes at 37 ° C, the reaction was stopped by adding oxilic acid. The absorbance at 405 nm was measured with a microplate electrometer.

3. Immunoblotting The antigen specificity of EMK-F7 and KHK-41 was analyzed by immunoblotting. Figure 1 shows Flow-2000, M of EMK-7 and KMK-41.
KN-45, MKN-28, MCF-7, KATO-III, MKN-45, KATO-III, MKN-45
Represents the immunoblotting pattern for (l
ane 1-8). MAb / KMN-41 reacted with the 56Kd band of each cell, but was oxidized by periodate to give MAb / KMK-41
The reactivity to KATO-III and KN-45 disappeared (lane 7-8).
MAb / EMK-F7 reacted with the 50Kd band of MKN-45, but the reactivity was lost by the periodate oxidation treatment. From this, it was revealed that MAb / EMK-7 recognizes a 56Kd glycoprotein on the surface of cancer cells.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, a human type monoclonal antibody that specifically recognizes the surface antigen of cancer cell of 56 Kd, and a hybridoma GM producing the human type monoclonal antibody
We offer KA, GMK-C3 or KMK-41. The human-type monoclonal antibody that specifically recognizes the 56 Kd surface antigen of cancer cells of the present invention specifically binds to various types of cancer cells, and thus this monoclonal antibody can be used to detect various types of cancer. Can be performed easily.

Furthermore, since the human monoclonal antibody that specifically recognizes the surface antigen of 56Kd of cancer cells of the present invention has very little immunogenicity to humans, it is useful as a therapeutic agent for various kinds of cancer. is there.

[Brief description of drawings]

FIG. 1 is an electropherogram showing immunoblotting of antigens sonicated by MAb / KMK-41 and MAb / EMK-F7 (supernatant) in various cell lines. Lane1 (Flow 2000), 2 (MKN-4
5), 3 (MKN-28), 4 (MCF-7), 5 (KATO-III), 6 (MKN-45), 7 (KATO
-III, periodic acid treatment), 8 (MKN-45; periodic acid treatment) is MAb
/ Reaction with KMK-41. Lane 9 (MKN-45), 10 (MKN-45; periodate treated) reacted with MAb / EMK-F7.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12N 15/07 // G01N 33/53 D 33/574 D 33/577 B (C12P 21/08 C12R) (1:91) (C12N 5/24 C12R 1:91)

Claims (5)

[Claims] 1. A humanized monoclonal antibody that specifically recognizes a 56 Kd surface antigen of cancer cells. 2. Hybridoma GMK-A, GMK-C3 or KMK
The human monoclonal antibody according to claim 1, which is produced by -41. 3. A hybridoma that produces a humanized monoclonal antibody that specifically recognizes a 56 Kd surface antigen of cancer cells. 4. The hybridoma according to claim 3, wherein the hybridoma is a human-human hybridoma. 5. The hybridoma is hybridoma GMK-A,
The hybridoma according to claim 3, which is GMK-C3 or KMK-41.