JPS60141285A - Mother cell strain for preparation of human hybridoma - Google Patents

Abstract

NEW MATERIAL:The mother cell strain for the preparation of human hybridoma which is a hypoxanthine-guanine-phosphoribosyltransferase-dificient mutant of a Namalwa cell which is a kind of human Burkitt's lymphoma cell. USE:A mother cell strain for the preparation of human hybridoma having excellent fusion capability to human lymphocyte B and capable of producing an antibody. PREPARATION:The cell is cultured in a serum medium added with preferably 30mug/ml of 6-thioguanine, at 37 deg.C in a 5% CO2 gas incubator, refreshing the medium every 3-5 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a parent cell line that has the ability to fuse with human B lymphocytes and is capable of producing human hybridomas with a high probability.

A feature of the method for producing monoclonal antibodies using hybridomas is that antibodies that react only with a single antigenic determinant can be repeatedly obtained in large quantities in vitro. Therefore, using this method, lI m .

It produces monoclonal antibodies against antigens such as viruses, and these antibodies can be applied to immunotherapy and biological/medical research.

Epstein-3 is a monoclonal antibody against the antigen.
Produced in culture of tile cells infected with apr virus.

Furthermore, Kohler et al. produced mouse monoclonal antibodies by cell fusion of mouse tile cells and mouse myeloma cells ( NatUrJ vol. 256, 495-4
97 Kai, 1975). Since then, many reports have been made regarding hybridomas between animal breast cancer cells and animal myeloma cells, but most of them are mouse hybridomas. However, since antibodies produced by mouse hybridomas are foreign to humans, there is a +1 risk of developing platform shock from repeated injections.

This has created a need for human monoclonal antibodies. To obtain human monoclonal antibodies, hybridomas of human B lymphocyte J, antibody-producing cells and permanently proliferative TR cells, such as human myeloma cells, are required.

However, human myeloma cells typically do not fuse well with human B lymphocyte cells to form hybridomas. Therefore, a human parent cell line with good fusion ability with human B lymphocytes has been desired.

On the other hand, when hybridomas are mass cultured in vitro to produce monoclonal antibodies, a medium supplemented with serum (hereinafter referred to as serum medium) such as fetal bovine serum (hereinafter referred to as FC3) has been used as the medium.

However, serum media are not suitable for large-scale culture because serum is expensive and there is variation between lots. Furthermore, purification of antibodies secreted into the culture medium is extremely complicated since the serum is added in multiple containers consisting of dozens or more components. Therefore, there has been a desire for a system in which hybridomas can proliferate and one monoclonal antibody can be produced even in J8 medium (hereinafter referred to as serum-free medium) without the addition of serum.

An object of the present invention is to provide a parent cell line that has an excellent ability to fuse with human B lymphocytes and, as a result, can produce human hybridomas that produce antibodies.

The novel parent cell line according to the present invention is Namalva (Namalva).
11
This is a defective strain of G P P T).

This parental cell line was named NΔT-30.

The Namalva cells used in the present invention are derived from a human B lymphocyte cell line and were derived from a patient with Burkitt's lymphoma. Namalva cells are widely available cells.

In the present invention, the selection of parent cell lines is as follows.

Hybridomas are selected from non-fused cells using a 24-well or 96-well plate, and the cells are dispensed so that one hybridoma is added or killed from each well. Therefore, hybridomas must be able to grow in large numbers of dead, non-hybrid cells. In many cases, even if normal parent cells undergo cell fusion, they do not proliferate after fusion.

For these reasons, the parent cell line used for fusion with human B lymphocytes is preferably a cell with a large and strong proliferative ability that can proliferate from one cell in one well.

As a method to obtain such cells, 1 out of 1 ul
Select cells that can proliferate from individual cells, and select cells that can proliferate in soft agar.

Cells that can grow even in a medium containing a large amount of dead cells are selected, and cells that grow in a serum-free medium are selected. The individual conditions for these selections and the number, order, etc. of these selection operations are changed as appropriate so as to obtain the desired cell line.

Note that the parent cells used for fusion with human B cells are preferably non-antibody-secreting cells. In this case, cells that do not secrete antibodies are selected from among the mutant Namalva cells.

This mutant cell of Namalva cells was 30Ij9/mQ
6-thioguanine (hereinafter referred to as 6-TG) at a concentration of
Further select for HGPRT deficiency in medium containing .

In this way, the l-I G P P T-deficient cells of the present invention are obtained.

The parent cell line NAT-30 obtained in this way is a new cell line that has not been described in any literature, and can be cultured permanently and stored frozen permanently.

The parent cell line of the present invention, NAT-30 cells, can be cultured in a variety of nutrient media. Nutrient media that can be used include, in addition to the commonly used serum medium (added with 10% FC8), for example, insulin 10#/me, transferrin 35
There is a surface-free medium (hereinafter referred to as ITES medium) in which four components, txt/m, 10 μM ethanolamine, and 2.5 x 10 M of S.L.
Volume 79.

1158-1162, 1982). As the basal medium, for example, DulbeCCO modified No. 8 base medium, RPM
I1640 medium etc. can be exemplified. 30% for each of the above media
It is preferable to add pg/- of 6-TG. still,
Normal Namalva cells do not grow well in 6TG supplemented medium. The culture conditions may be those used for normal cell culture.

Generally, cells can be grown well by culturing in a 5% carbon dioxide incubator at about 37° C. and replacing the medium every about 3 to 5 days.

The NAT-30 cells of the present invention can be used as parent cells for cell fusion with human B lymphocytes. Human B fil+ cells that can be used in Bm cell fusion are not particularly limited, and include, for example, B cells derived from lymph nodes, spleen, peripheral blood, and the like. These B111 cells can be isolated and purified by various commonly used separation means and subjected to cell fusion with the parent cells of the present invention.

The fusion reaction between the NAT-30 cells of the present invention and the human B cells described above is basically the same as known cell fusion methods, and is carried out in an appropriate medium in the presence of a fusion promoter.

As a fusion promoter, for example, polyethylene glycol (
(hereinafter referred to as PEG) can be advantageously used. PEG preferably has an average molecular weight of about 1,000 to 2,000, and is suitably present in the medium at a concentration of 30 to 50% (W/'V). In addition, the medium includes the above-mentioned basal medium used for proliferation of NAT-30 cells, and Dulbecco's modified medium.

Various conventional media such as RPM I 1640 medium can be used.

Furthermore, an adjuvant such as dimethyl sulfoxide may be added to the cell H11 culture medium to increase the fusion efficiency. The cell number ratio of NAT-30 cells and human B lymphocytes used for cell fusion is usually
Approximately 1 to 5 times as many human B cells as cells are used.

Cell matching is performed, for example, as follows. That is, NAT-30 cells and 1 to B cells are mixed in an appropriate medium, centrifuged,
A PFG solution heated to 37° C. is added to the obtained cell pellet and mixed. Thereafter, an appropriate medium is added little by little, centrifuged, and a normal selection medium is added to the resulting cell pellet to separate the desired fused cells. id
A separate medium is a medium in which the parent cells are killed and only the target hybridoma can grow, and a typical example is a HAT medium.

As the HAT medium, for example, 15% FC8-containing D
Hyboxanthin 1×1 in u + b6cco modified medium
A 18-molecular solution supplemented with 0-cow M, aminopterin 1 xio-'7 M and demidine 1.6 x 10-5 M is used.

Cells are cultured in the 1" hepatoma medium for about 2 to 4 weeks, which is sufficient to kill cells other than the target hybridoma. This selectively proliferates only the target human B cell hybridoma. J to l-IAT medium
After selection, the cells are cultured for about one week in a medium containing hyboxanthin and thymidine, and then cultured in a normal medium.

Hybridomas generated by the fusion of the NAT-30 cells of the present invention and human B lymphocytes are 6 to 4 weeks later.
62 out of 68 wells (1×10” NAT-3
0 cells followed by 1 hybridoma). The appearance frequency of this hybridoma is based on the conventional one by Cote et al. (Proc, Natl, Acad, Sci, USA
, Vol. 110, pp. 2026-2030, 1983)
several times higher. Growing in HAT medium means that N
Figure 2 shows successful fusion between AT-30 cells and human B cells. These cells were grown continuously in l-IAT medium and cloned by limited dilution. The NAT-30 cells used are non-antibody-secreting cells, but
Approximately 24% of the cloned hybridomas secreted I (IG or IoM) and the number of chromosomes in NAT-30 cells was 46±2, whereas the number of chromosomes in one hybridoma clone was 57±1. and was clearly increasing.

As described above, the NAT-,30 cells of the present invention proliferate in a serum-free medium (for example, ITES medium) in the same manner as in a serum medium. Hybridomas between NAT-30 cells and human B cells can also sufficiently proliferate in serum-free media such as ITES medium and produce antibodies. That is, when purifying antibodies secreted from a medium by mass culture of hybridomas, if NAT-30 cells are used as parent cells, compared to conventional purification of antibodies from serum medium, it is possible to purify antibodies without serum. It also becomes possible to purify antibodies very easily and inexpensively using a medium.

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

Example 1 - Production of 1-I G P P T-deficient human I hennamalva mutant cell line First, Namalva cells (Dainippon Pharmaceutical Co., Ltd.) were incubated at 45°C with a concentration of 0.25
5% Dulbecco's modified medium containing agar + 10 modified medium + 1 medium was placed in a 50 m petri dish and cultured for 3 weeks at 37° C. in an incubator with 5% carbon dioxide gas and 95% air. Thereafter, 90 clones that had grown were taken out one by one into 96 wells (200 p of medium per well) and similarly cultured for 2 weeks in the above serum medium without agar. For the purpose of selecting strains incapable of producing antibodies, the culture supernatant from each well was taken and the antibody n in the supernatant was measured using an enzyme immunoassay (Cabbell). As a result, seven cell lines in which no antibodies were detected were selected and sufficiently grown in 96 wells. Proliferated cell lines were placed in 24 wells each (1.
5 mQ medium) and 5 cm Petri dishes (5 d medium per plate) to increase the amount of culture solution until approximately 5 x 1
06 cells were obtained. 3 x 104 for each stock
The cells were left and the other cells were suspended in 15 m2 of the above freshly prepared serum medium, and the cells were killed by repeating the freezing/thawing operation twice by freezing at -5°C and then thawing at room temperature.

Transfer 5 mQ of the culture medium containing these dead cells to three 50 m Petri dishes, and add 1x each of the cells that remained unkilled.
104 cells each were transplanted. 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 each strain was transplanted into 30 wells of a 96-well plate. As a result of culturing for 3 weeks while replacing the medium every 4 to 6 days, the strains that showed proliferation were the 7 strains mentioned above.
It was about 2 of the stocks. For the two strains, the culture medium volume was increased in the above order for cells in five wells each having a fast proliferation rate, and approximately 1 x 106 cells were obtained in each well. Each cell was collected separately by centrifugation and once Du + becco
Wash with modified medium and serum-free medium (ITES medium)
Each was suspended at 5m (!).

Culture was carried out in the same manner as above for 4 weeks, changing the medium every 4 to 6 days. 3 from the fastest growth rate when cultured in this serum-free medium
A type of cell was selected and sufficiently proliferated to obtain about 1 x 10 cells each. Each of these was added to 10 d of the above serum medium containing 3 μg/me of 6-TG.
and cultured for 4 weeks while replacing the medium every 4 days. Then, viable cells were removed and 30 μg/- of 6-TG was added.
The cells were suspended in the same medium containing 1 x 10'' cells/me iE, and cultured for 4 weeks in the same manner as above.

In this way, two 6-TG resistant strains were obtained. When these two strains were then subjected to cell fusion by the method described in Example 2 below, one of the two strains showed particularly strong fusion ability. This strain was named NAT-30. This stock is 3
011! / m (!) It showed strong growth in the above serum medium containing 6-TG and a supernatant-free medium, and was maintained in these medium for subculture.

Example 2 Cell fusion between NAT-30 cells and human B lymphocytes Cell fusion was performed using the NAT-30 cells obtained in Example 1. The day before fusion, the parent cells NAT-30 were prepared using 10% FC8-containing Duul Becco modified medium.
c + g ta L/, a state of active proliferation was achieved.

Human B lymphocytes were obtained by cutting a lymph node excised from a cancer patient into small pieces in the above medium and removing lymph cells. This was cultured for 1 hour in a 5% carbon dioxide gas incubator at 37°C, and the human B lymphocytes were removed by removing macrophages that had adhered to the shell.

3X 10'' of the above NΔT-30 cells and the above human hB
1×10 g of lymphocyte cells were used for cell fusion.

Wash each cell 1) twice with ulhccco modified medium and
Mix in a 0 m centrifuge tube at 1.20 Or, p, m, 7
Centrifuged for minutes. The supernatant was completely removed and 1me of 42.5% PFG (average molecular weight 1.500) and 15% 5% dimethyl sulfoxide Dulbecco's modified medium warmed to 31°C was added in small portions to the resulting cell pellet. Ta.

After shaking gently for 1 minute, Dulbec was warmed to 37°C.
c.

As is clear from N A T -30 and X 63
, 6.5.3, both IgG and IoM producing types were observed. In addition, human IoG and I(IM) assays were performed using the enzyme immunoassay described in Example 1.

Cultured NAT-30 cells were suspended in the above serum-free medium (TTES medium) and 10% FC8% at a cell concentration of 2 x 10 cells/d, and 2 mQ each was placed in a 3.5 cm Petri dish and incubated at 37°C. The cells were cultured in a 5% carbon dioxide incocovator. The number of cells was measured using a Rel Counter (Toa Medical Electronics Co., Ltd.). As is clear from FIG. 1, the sea urchins showed good growth even in serum-free medium. In Fig. 1, Δ represents the increase in rJ in ITES medium.
M indicates growth in serum medium. In addition, the serum-free medium was replaced every 3 to 5 days.
Since it has been possible to propagate 0 cells well and to continuously expand them, it is possible to carry out permanent proliferation in a serum-free medium.

Example 5 Hybridoma 1 in serum-free medium 1-IF10B4, one of the hybridoma clones, was cultured in serum-free culture under the same conditions as in Example 4. As shown by the growth curve in FIG. 2, this hybridoma grows well in serum-free medium, and its growth in the logarithmic phase is almost the same as that in serum medium. Moreover, this hybridoma is persistent even in serum-free medium (!
It is possible to do 1m. In addition, in Figure 2, △ is r
Growth in TEs medium is shown, and Mu shows growth in serum medium.

Example 6 Several serum-free hybridoma clones were cultured under the same conditions as in Example 4, and antibody production ability, 5
The cell density reached at 8th point and the doubling time in the logarithmic growth phase were determined. Each clone was incubated with IOG or T in serum medium.
(Two strains for IgG were randomly selected from among the clones producing IM.

1 (For IM, 5 strains were selected for the experiment. As is clear from Table 3, serum-free medium has the same I
Each clone produces qG or IaM, and the cell density reached on day 5 is approximately the same. The doubling time for logarithmic increase in TAItI was almost the same when serum-free medium was used as when serum medium was used. Therefore, N.A.
If T-30 cells are used as parent cells, hybridomas that grow in a serum-free medium and produce antibodies can be obtained.

Table 3 A H.B. H.B. HF HF+ HF8D11-+11 21 26.6 NTF1 -IF T

[Brief explanation of the drawing]

Figures 1 and 2 show NAT-30 cells and hybridoma clone l-IF 10B4, respectively.
FIG. Δ...I TFS medium, Mu... Serum medium. Attorney Jō Imamura Figure 2

Claims (1)

[Scope of Claims] (1) A parent cell line for producing human hybridomas, which is a hypoxandine-guanine-phosphoribosyltransferase-deficient mutant cell of Namalva cells, which are human Burkitt's lymphoma cells. (T) The parent cell line according to claim 1, which is characterized in that it does not produce antibodies. Parent cell line according to item 2. (4) Any one of claims 1 to 3, characterized in that it has the ability to fuse with antibody-producing cells (human B lymphocyte cells). The parental cell line described in.