JPS61242574A - Ouabain-resistant cell derived from human burkitt's lymphoma - Google Patents

Abstract

PURPOSE:To obtain a hyridoma capable of multiplying permanently a specific monoclonal antibody, by imparting ouabain resistance artificially to a fusible cell derived from human Burkitt's lymphomas. CONSTITUTION:A parent cell for imparting ouabain resistance is obtained by separating a cell capable of fusing with a cell producing an antibody in lymphoblasts separated from human Burkitt's lymphomas. For example, a cell designated as NAT-30 is used as the above-mentioned cell. The NAT-30 can be fused with a cell producing the antibody derived from humans, and has sensitivity to aminoputerine having 4X10<-7>M and further sensitivity to HAT culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to cells derived from human Burkitt's lymphoma and artificially endowed with ouabain resistance.

[Conventional technology]

Cell fusion technology [Nature, 25
6.495 (1975)), various monoclonal antibodies have been produced by applying this technique.

This technique generally involves fusing tumor cells and antibody-producing cells in the presence of a cell fusion agent, and then obtaining subcultureable cells (hybridomas) that produce the desired monoclonal antibody (for example, , Japanese Patent Application Publication No. 1983-
29622, etc.). To create human monoclonal antibodies using this method, (1) mouse,
A method for obtaining a hybridoma by fusing a myeloma cell line from an experimental animal such as a rat with a human type antibody-producing cell, and culturing this to obtain a desired antibody, and (2) a human myeloma cell line and a human type. There is a method of obtaining a hybridoma by fusing it with an antibody-producing cell of 1, and culturing the hybridoma to obtain the desired antibody. However, method (I) has the problem that the human chromosomes in the obtained hybridomas are shed over time and lose the ability to produce antibodies, making it extremely difficult to maintain the ability to produce antibodies. Furthermore, method (2) has the problem that the desired monoclonal antibody cannot be sufficiently supplied because the cell fusion efficiency is low and the types of cells that can be used for fusion are also limited.

Note that without applying cell fusion technology, antibody-producing human B lymphocytes were obtained according to a conventional method, and these were infected with Epstein-Barr Virus (EB
V)) can be used to establish an antibody-producing strain against a specific antigen and obtain the desired antibody.
Generally, such antibody-producing strains are difficult to clone and have problems such as rapid decline in antibody production ability.

Therefore, in order to deal with these problems, we first transformed human B lymphocytes with EBV [Nature
re), 269.420 (1977)), once an antibody-producing strain against a specific antigen is established, this strain is fused with human tumor cells, which is more stable than the original cell line and produces antibodies in large quantities. Methods for producing the resulting hybridomas can be considered. However, in this way EBV
Since antibody-producing cells produced by transformation with 11/IT medium can also proliferate in II/IT medium, such antibody-producing cells and hybridomas cannot be distinguished in 11/IT medium by conventional methods.

Therefore, in order to obtain the desired hybridoma in such cases, cells into which a new marker has been introduced are required as tumor cells to be fused with the antibody-producing cells transformed with the RBV, and currently such cells are not available. It is hoped that the provision of

[Problem that the invention seeks to solve]

Therefore, the present invention seeks to provide tumor cells into which a marker capable of distinguishing between antibody-producing cells transformed with EBV and hybridomas formed by cell fusion of the antibody-producing cells and tumor cells is introduced. It is something to do.

[Means for solving problems]

The above problems are derived from the following properties: (])
2) Resistant to ouabain; (3)
The problem is solved by providing a cell which can fuse with an antibody-producing cell transformed by IEIIV.

[Specific explanation]

Generally, the partner cell for the fusion of antibody-producing cells to obtain a hybridoma that can proliferate permanently and produce a specific monoclonal antibody is itself capable of proliferating permanently; It must not produce the specific antibody alone and must be able to fuse with antibody-producing cells. Furthermore, the partner cell for the fusion of the antibody-producing cell must have a marker that the antibody-producing cell does not have, allowing discrimination between the antibody-producing cell and the hybridoma to which the marker has been transferred. Must be. In order to obtain cells that do not meet these conditions, in the present invention, ouabain resistance is artificially imparted to fusogenic cells derived from human Burkitt's lymphoma.

The parent cells that confer ouabain resistance can be obtained, for example, by isolating cells that can fuse with antibody-producing cells from lymphoblasts isolated from human Burkitt's lymphoma. can get.

As such a cell, for example, a cell named NAT-30 can be used, and this cell has the following properties.

(11 Can be fused with human-derived antibody-producing cells; (Sensitive to 214-xlO-7M aminopterin; Sensitive to 431 11 AT medium.

NAT-30 cells can be constructed as follows. First, human Burkitt's lymphoma cells were grown in a 6-thioguanine (6-TG)-containing medium (RPMI-1640 medium; Dulbecco's modified MEM medium: F12 medium - 2:1:1).
(containing 10% beef tallow serum ((Fe2)))], and the concentration of 6-TG was gradually increased until the final concentration of 6-TG was 30.
Obtain cells that grow in μg/ml containing medium. Note that this specific method will be described in detail in Example 1.

Creation of ouabain-resistant cells The ouabain-resistant cells of the present invention are produced by (A) inducing cell mutations by treating the above-mentioned fusion-capable cells with a mutagen, (B) selecting mutated clones, (C) Can be obtained by further isolation of single clones. Preferably, the stability of the established cells is checked. Such a series of operations is well known and can be referred to in various books and documents [“Practical Manual for Utilizing Animal Cells” P219-228]
(19B'4), published by Realize Publishing.

(A) Mutagen Drugs are generally used as cell mutagens, such as N-
Methyl-N'nitro-N-2-rosoguanicine (MN
NG), ethyl methanesulfonate (IEMs), IC
R'-191 etc. are used. The type of mutagen, its usage time, usage concentration, and other usage conditions can be appropriately selected depending on the cells to be mutated. In the present invention, it is preferable to use ICR-191, and this ICR-191 is preferably used.
CR491 is 6-chloro-9-〇-(3-(2-chloroethylamino-propyl)amino)-2-methoxyacridine dihydrochloride (6-chloro-9-o-
(3-(2-chloroethylamino)-
pro-pyl)amino)-2-Wlethoxy
acrydine dihydrochloride)
and is available from Polyscience.

The mutation process can be performed, for example, as follows.

When the parent cells, e.g. NAT-30 cells, are grown in a medium in which they can grow and reach logarithmic growth phase,
A mutagen, such as ICR-191, is added to a predetermined concentration, preferably at multiple levels, and the culture is continued. When the cell concentration reaches a predetermined concentration, for example, about 1×10 6 cells/ml, the cells are collected by centrifugation, diluted and suspended to about 1/10 of the concentration in the same medium containing no mutagen, and cultured again. Repeat this multiple times. The mutagenized cells are then selected as described below.

(B) Selection of mutant clones and separation of single clones Selection of mutant clones is carried out by culturing the mutated cells as described above in a medium containing a predetermined concentration of ouabain, and selecting growing cells. conduct.

After collecting the cells obtained above, the cells are cultured and further cultured in a medium supplemented with ouabain in the same manner as above to obtain growing cells to isolate a single clone.

Characteristics of ouabain-resistant cells Ouabain-resistant NAT-30 cells, which are an example of the ouabain-resistant cells of the present invention, are obtained by imparting ouabain resistance to parent cells, NAT-30 cells, and have the following properties.

(11) can be fused with antibody-producing cells, such as human-derived EBV-transformed antibody-producing cells, to form hybridomas capable of persistent proliferation and stable production of monoclonal antibodies. - f210.
It can proliferate in the presence of 1 μg/ml of ouabain, and its resistance to ouabain is about 10 times greater than that of the parent cell, NAT-30 cells.

(3t 4xlO-'M sensitive to aminopterin.

(Sensitive to 41HA T medium.

Furthermore, it has the following culture characteristics.

(51' The growth rate in RPMT-1640 medium containing 10% fetal bovine serum (Fe2) is a cell doubling time (PDT) of 15 to 20 hours.

(61IMl'1M medium (containing 10% FC3) proliferates well, IM leaf medium (20% FSC, 10% DMSO (
dimethyl sulfoxide] is suitable as a preservation-like freezing medium.

(7) Sufficient cloning can be achieved without adding feeder cells.

〔Effect of the invention〕

The cells of the present invention are tumor cells that have ouabain resistance. Therefore, it is possible to transform human B lymphocytes with EBV and establish a monoclonal antibody-producing strain against a specific antigen, and then to fuse these cells with the ouabain-resistant cells of the present invention to subsequently select desired antibody-producing cells. Become. That is, by adding ouabain to a selective medium for desired antibody-producing cells, desired antibody-producing cells can be efficiently obtained.

Next, the present invention will be explained in more detail with reference to Examples.

[Example], Creation of NAT-30 cells First, Namalva cells (Dainippon Pharmaceutical Co., Ltd.) were grown at 45°C in a Dalheco modified medium containing 0.25% agar + serum medium supplemented with 10% FC8 1×1.
03 pieces/mI! Cells were suspended at a concentration of
% air in an incubator for 3 weeks. Thereafter, 90 grown clones were taken out one by one into 96 wells (200 μβ medium per well) and similarly cultured for 2 weeks in the above serum medium containing no agar. For the purpose of selecting strains incapable of producing antibodies, culture supernatants from each well were collected and subjected to enzyme immunoassay (Kappel).
The amount of antibody in the supernatant was measured. As a result, seven cell lines in which no antibodies were detected were selected and sufficiently grown in 96 wells. Each grown cell line was placed in 24 wells (1.5 mβ medium per well), 5C
The volume of culture solution was increased by using 11 plates (5 ml of medium per plate) in order to obtain about 5 x 1.06 cells each. For each strain, 3 x 10' cells were left and other cells were suspended in the freshly prepared serum medium i5mA, and the cells were killed by repeating the freezing/thawing operation twice by freezing at -5°C and thawing at room temperature. . 5 mff of the medium containing the dead cells was transferred to three 5 cm petri dishes, and the remaining unkilled cells were transplanted into each dish at 1×10′ cells. When cultured for 5 days in the same manner as above, most of the cells died, but the surviving cells were collected by centrifugation and transplanted into 30 wells of a 96-well plate for each strain (I2).

As a result of culturing for 3 weeks while replacing the medium every 4 to 6 days, 2 of the 7 strains were observed to proliferate. 2
Regarding the strain, the culture amount of 5 frog cells was increased in the above order at a rate of growth of 5 degrees, and approximately 1X10' cells were obtained for each. Collect each cell separately by centrifugation,
The cells were washed twice with Dulbecco's improved medium and suspended in 5 mρ of serum-free medium (rTEs medium). Culture was carried out in the same manner as above for 4 weeks, changing the medium every 4 to 6 days. Three types of cells were selected based on the fastest proliferation rate when cultured in this serum-free medium.
The cells were sufficiently grown to obtain about 1×10 7 cells each. Each of these is 3μg/mj! 10m7 of the above serum medium containing 6-TG! and cultured for 4 weeks while replacing the medium every 4 days. Then, remove viable cells and add 30μ
3 lXIO/in the same medium containing g/ml 6-TG.
m7! ? ! Float it so that it is 1 degree, and repeat 4 times as above.
Cultured for a week. In this way, two 6-TG resistant strains were obtained. This strain was named NAT-30. This strain has 30μg/m7! It shows strong growth in the above-mentioned serum medium containing 6-TG and serum-free medium, and is maintained by subculture in these media.

101 cases “Synthesis of euabain facial NAT-30 cells”
The NAT-30 cells generated as described above were added to 20 m #03 Tsuno TMDM medium (containing 10% FC3) (Boehringer Mannheim) at 5 x 10' cells/m.
The cells were inoculated at a concentration of 1 and cultured at 37°C and 5% CO2.

When the culture reached logarithmic growth phase (2X 1.O' cells/m
R1 total 4 x 106 cells), and the mutagen ICR-191 (Polyscience) was added to each culture at a final concentration of 0.5 μg/mj! , 1. Opg/ml, or 2.
It was added at a concentration of 0 μg/mjH, and the cells were further cultured for 15 hours. After culturing, collect the cells by centrifugation and add 2xI to a new ouabain-free medium with the same composition as above.
O' cells/mj2 were suspended and cultured under the same conditions as above. Every time the cell concentration reached approximately 1×10' cells/ml, ouabain-free medium of the same composition was added to dilute the mixture to a cell concentration of approximately 1×10 5 cells/ml, and the culture was continued. Cultivation was carried out in this manner for 10 days. The growth state of the cells in this case is shown in FIG. Total cell number (cells) on vertical axis
and the number of culture days is shown on the horizontal axis. In the figure -〇-1-△-1
and -x- indicate the final concentration of ICR-191 as 0.
5μs/mj! .

1, Op g /mA, and 2.0 p g /m
Indicates the growth state of cells treated as j+, -・- is r
The growth status of cells not treated with cR-191 is shown. The arrow in the figure indicates the day on which the dilution was performed, and the numerical value above the arrow indicates the dilution rate.

Next, the cells obtained as above and treated with 0.5 μg/ml of ICR-191 were collected, and the cells were treated with 0.03 μg/ml of ouabain, respectively. ! , 3μg/mj2
, and IMDM medium containing 30 pg/r+l (
(containing 10% FC5) and distributed into two 96-well plates for each ouabain concentration. In this case, the number of cells was 10' cells/well.

This was cultured for 7 days. As a result, the ouabain concentration was 0.
03μg/rl! On the other hand, cell proliferation was observed for all types of ouabain on the ouabain plate.
/ml! In the plate, cell proliferation was observed in three wells, and the ouabain concentration was 3 μg/mI! and 30μg
/ m I! No cell proliferation was observed. Therefore, cells obtained on a plate with an ouabain concentration of 0.3 μg/me were obtained as ouabain-resistant NAT-30 cells. Ouabain-resistant NAT-30 obtained here
FIG. 2 shows the growth state of cells in IMDM medium containing ouabain (containing 10% FC3).

In the figure, -.- indicates the growth state of NAT-30 cells as a control, and -0- indicates the growth state of ouabain-resistant NAT-30 cells. Furthermore, the numbers in parentheses indicate the concentration (M) of ouabain added to the medium. From this result, the ouabain-resistant NAT-30 cells established in the present invention are
It was found that the cells were resistant to ouabain at a concentration about 10 times higher than that of T-30 cells.

This was sensitized in vitro with Pseudomonas aeruginosa F7 (Fisher type 7), and then the B lymphocytes were transformed with EBV to select cells that produced antibodies against Pseudomonas aeruginosa (
The method for preparing these cells is described in detail in Japanese Patent Application No. 59-212579). These antibody-producing cells and the ouabain-resistant NAT-30 cells obtained in Example 2 were used in a conventional manner [
When clones producing antibodies against Pseudomonas aeruginosa F7 were selected, 3 out of 96 wells produced the desired antibodies.

[Brief explanation of drawings]

FIG. 1 is a graph showing the growth state of NAT-30 cells in a TCP-191-containing medium. FIG. 2 is a graph showing an example of the growth state of cells according to the present invention in an ouabain-containing medium. Figure 1 Figure 2-9A-

Claims (1)

[Claims] 1. The following properties: (1) Derived from human Burkitt's lymphoma; (2) Resistant to vain; (3) Antibody production transformed by Epstein-Barr virus. A cell that is capable of fusing with a cell; 2. In addition, the following properties: (4) sensitive to aminopterin; (5) H
The cell according to claim 1, which is a vain-resistant NAT-30 cell having: sensitive to AT medium.