JP3084030B2 - New hybridoma - Google Patents

Description

The present invention relates to a novel hybridoma.

[Problems to be solved by conventional technology and invention]

Monoclonal antibody by the cell fusion method developed by Koehler and Milstein (hereinafter referred to as "MoAb")
Is a method of fusing mouse myeloma cells with mouse acidic lymphocytes, and a method for obtaining mouse MoAb by selecting only fused cells of both cells is widely used. Antibodies produced by these fused cells show high specificity for one type of antigenic determinant and can be obtained with good reproducibility as needed, so they are widely used for research, in vitro diagnostics, purification of bioactive substances, etc. Used.

However, since this type of mouse MoAb is composed of mouse globulin, it is a heterologous protein for humans, and when administered to humans, there is a risk that sometimes severe shock symptoms may occur. Various attempts have been made to avoid this problem and to develop MoAbs as in-vivo diagnostics and therapeutics. Among them, a method for producing human-type MoAbs by applying cell fusion to human lymphocytes is promising.

Conventionally, the following method has been used to produce human MoAb. That is, (1) a method using a human-human hybridoma, (2) a method using cells transformed with Epstein Barr Virus, (3) a method using a human-mouse-heterohybridoma, and the like have been reported. ing. However, in the case of (1), the fusion efficiency is low, in the case of (2), cloning is difficult, and in the case of (3), the human chromosome is liable to drop out, and stable human antibody production is not achieved. There were drawbacks such as difficulty in obtaining. For the purpose of improving (3), for example, a hybridoma consisting of human lymphoid cells and a lymphoid cell line of a heterologous animal (JP-A-61-128885)
No.) has been reported, but there have been problems such as the proliferation of the human-mouse heterohybridoma parent strain and the efficiency of fusion with the antibody-producing cells being poor, and the actual situation has not been achieved yet.

[Means for solving the problem]

Under such circumstances, the present inventors have conducted intensive studies for the purpose of providing useful monoclonal antibodies by fusing human-mouse heterohybridomas with human antibody-producing cells. As the human-mouse heterohybridomas to be used, those obtained from human fetal spleen cells and having 66 to 70 chromosomes per cell, have extremely high fusion efficiency and cloning efficiency, and stably maintain human-type immunity after fusion. The present inventors have found that globulin (hereinafter, referred to as “Ig”) can be produced, and have completed the present invention.

That is, the present invention relates to an 8-azaguanine-resistant human clone obtained by cell fusion between human fetal spleen cells and mouse myeloma cells NS-1 and having 66 to 70 chromosomes per cell. The present invention provides a mouse heterohybridoma.

The human-mouse heterohybridoma of the present invention is produced, for example, by fusing human fetal spleen cells with mouse myeloma cells NS-1 and making the resulting fused cells resistant to 8-azaguanine.

Cell fusion is usually performed using human fetal spleen cells and mouse myeloma cells in a medium such as MEM medium, RPMI1640 medium, or IMDM medium.
This is carried out by mixing NS-1 (mixing ratio is usually 10: 1 to 5: 1). As a fusion promoter, the average molecular weight is 1,000 ~
6,000 polyethylene glycols (PEG) can be used.
The working concentration of PEG is usually 30-50%. Heterohybridomas can be obtained by culturing the cells after cell fusion in a selection medium (for example, hypoxanthine-aminopterin-thymidine (HAT) medium).

The resistance to 8-azaguanine (hereinafter referred to as "8AG") is obtained by culturing the heterohybridomas obtained as described above in an RPMI1640 medium containing 10% FCS containing 8AG. That is, the final concentration of 8AG is 100μ
Culture is performed by gradually increasing the concentration of 8AG until the concentration reaches about M.

If the obtained 8AG-resistant hybridoma is cloned, for example, by a limiting dilution method, a desired human-mouse heterohybridoma can be obtained as a clone.

Mouse and human heterohybridomas 2A-5, 4D-5, which have a high fusion efficiency with human lymphocytes
11E-5 was obtained. Each of these hybridomas is a novel cell, and among them, 11E-5 was deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology (Microorganisms No. 11094 (FERM P-11094)).

Based on the fusion efficiency with human antibody-producing cells, the growth potential of the hybridoma, the ease of cloning, and the ability to produce antibodies, the obtained heterohybridoma should be selected and established as a heterobryoma useful as a parent strain. preferable.

When this heterohybridoma is used as a parent strain when producing a human-mouse-human triple hybridoma, the fusion efficiency and the cloning efficiency are high. Further, the parent strain itself does not produce Ig, and is characterized by stably producing Ig after fusion.

〔Example〕

 Next, the present invention will be further described with reference to examples.

Example 1 (1) Production of 8-azaguanine resistant human-mouse heterohybridoma parent strain: Lymphocyte layer was separated from human fetal spleen by Ficoll-back (Pharmacia) specific gravity centrifugation, and RPMI1640 medium (Gibco). After washing three times, spleen cells were obtained.

The separated spleen cells and 10% fetal bovine serum (hereinafter, referred to as
PEG4000 (manufactured by Sigma), which was cultured in an RPMI1640 medium containing “FCS” and mixed in a logarithmic growth phase with mouse myeloma cells NS-1 at a cell ratio of 10: 1 or 5: 1, and previously kept at 37 ° C.
Was added and the mixture was allowed to stand for 1 minute. 30 ml of this cell mixture
RPMI1640 medium was added in small portions over 10 minutes to dilute the PEG. After dilution, the cells were precipitated by centrifugation at 400 × g for 3 minutes, and adjusted to 1 × 10 ⁶ cells / ml with RPMI1640 medium containing 10% FCS. Transfer the cell suspension to a 96-well microplate for 10 minutes.
The cells were dispensed by 0 μl and cultured. The next day, RPMI16 containing 10% FCS
HAT medium, which was obtained by adding HAT to 40 medium, was added at 100 μl per well.

Thereafter, every 100% of the medium was removed from the wells every 3 to 4 days, and a fresh 100 μM HAT medium was added. Three weeks later,
Cell proliferation was observed in approximately 3.4% of the wells. Among them, the strain that produced Ig was selected and named II strain.

Then, the II strain was subcultured in HAT medium for 2 weeks, and then
After subculture for 2 weeks in RPMI1640 medium containing 0% FCS, cloning was performed, and after 8 weeks, each clone was obtained. The II strain cloned in this manner was cultured in an RPMI1640 medium containing 10% FCS to which 8-azaguanine (8AG) having a final concentration of 2 μM was added, and thereafter, 4, 8, 16, 32, 50, and 100 μM of 8AG were added. The cells were cultured in an RPMI1640 medium containing 10% FCS, and resistance to 8AG was measured sequentially. After about two months, a final concentration of 100 μM 8
Cells growing in AG-containing medium became evident. The proliferating cells were cloned by limiting dilution using peritoneal cells of BALB / c mice as feeder cells, and 8AG-resistant human-mouse heterohybridomas were established.

The established cells can be grown in a medium containing 8AG,
In the AT medium, all cells were killed after 4 days of culture, confirming that the cells were sensitive to HAT. Fig. 1 shows the
It showed growth in the medium containing 8AG and the HAT medium.

Furthermore, measurement by enzyme immunoassay revealed that it was a non-secretory form of human immunoglobulin and mouse immunoglobulin. In addition, the result of chromosome analysis (CRAc
ad. Sc. (paris) 276, 3179 (1973)), and these cells were found to be heterohybridomas harboring both human and mouse chromosomes.

(2) Examination of fusion efficiency: (i) Fusion with mouse lymphocytes Lymphocytes (2 × 10 ^{7 /} ml, 0.4
ml) and 33 clones (2 ×
(10 ^{7 /} ml, 0.4 ml) was subjected to cell fusion by the electric pulse fusion method, and the fusion efficiency of each clone was compared based on the number of wells that were seeded in 96 wells and grown in HAT medium. The results are shown in Table 1.

Before the cloning, the II strain showed cell growth in 37 of 96 wells. On the other hand, each of the 33 clones formed a hybridoma of 12 to 96 wells. Among them, 5 clones of 2A-5, 2H-5, 4D-5, 6D-5, and 11E-5 having a high hybridoma formation rate and having 66 to 70 chromosomes per cell were selected.

(Ii) Fusion with human lymphocytes Lymphocytes (2 × 10 ^{7 /} ml, 0.4 ml) isolated from normal human peripheral blood by the Ficoll method and 5 clones which showed high fusion efficiency with mouse lymphocytes were used for electrophoresis. Pulse fusion. The fusion efficiency of each clone with human lymphocytes is shown in Table 2.

All 3 clones of 4D-5, 11E-5 and 11A-6 were 96
Hybridomas were formed in more than 90 wells.

Reference Example 1 Production of human Ig by triple hybridoma: Lymphocytes separated from normal human peripheral blood by Ficoll method and mouse-human heterohybridoma (II strain) were subjected to cell fusion by PEG and electric pulse according to a conventional method. . Table 3 shows the fusion efficiency and the appearance rate of Ig-producing cells.

In either fusion method, triple hybridomas lymphocytes 10 ⁶ per three or more, occurrences of Ig producing cells
The value was as high as 0.3 or more.

Reference Example 2 Properties of Triple Hybridoma: The doubling time and MoAb production state of the triple hybridoma producing MoAb obtained in Reference Example 1 were examined. The results are shown in Table 4.

The doubling time in the hypoxanthine-thymidine (HT) -supplemented medium and the serum-free medium was as wide as 22 to 36 hours by the triple hybridoma. MoAb production amount of production 10 ⁶ cells over a period of 8 months or more of long-term per day 2μg or more, and secretion.

Test Example 1 DNA analysis of heterohybridoma: Method of Maniatis et al. (Molecular Cloning, Cold spring) from heterohybridoma II strain and mouse myeloma cells
DNA was extracted according to Harbor laboratory NY, 1982, p280, and this was extracted using the method of Arakawa et al. (Chemother., 33 , 63 (198
9)) and Southern blot hybridization was performed. The DNA was cut with Eco R1, Hind III and Bam HI restriction enzymes, electrophoresed on a 0.9% agarose gel, and transferred to a nitrocellulose membrane by the Southern method (J. Mol. Biol., 98 , 503 (1975)). Transcribed. Subsequently, the transferred membrane was labeled with ³² P-labeled Blu8 containing human specific Alu DNA.
Were reacted with each other to detect DNA complementary to human DNA. As a result, it was proved that the heterohybridoma II strain contained human DNA as shown in FIG. Also, a triple hybridoma (C8) 11B-F obtained by fusing a heterohybridoma II strain with human lymphocytes
It was confirmed that the six strains also contained human-derived DNA.

Test Example 2 Chromosome analysis: The chromosome analysis of the fused cells was performed by the BrdU-AO method (CRAcad.Sc.
(Paris) 276, 3179 (1973)). That is, cells were cultured for 9 hours in a medium supplemented with 100 μg / ml of 5-bromodeoxyuridine (BrdU), followed by colcemide.
Take out the cells cultured for 20 minutes after adding 0.05 μg / ml,
The mixture was fixed with a 3: 1 mixture of methanol and acetic acid. The fixed cells were fixed on a slide glass, stained with 0.13 mg / ml of acridine orange (AO) solution (pH 7.0) for 15 minutes, and then chromosomes were observed with a fluorescence microscope.

As a result, the heterohybridoma II strain had 54 chromosomes.
It was distributed in 本 75 and averaged 67. Of these, 21
When chromosome analysis was performed on individual cells, 2 to 7 human-derived chromosomes were included. In addition, the presence of human chromosomes Nos. 3, 12, 16, 17, and 19 was observed in more than half of the cells.

On the other hand, the number of chromosomes of the triple hybridoma (C8) 11B-F6 strain was distributed from 58 to 84, with an average of 76.

Chromosome analysis of 20 cells revealed that 3 to 16 human-derived chromosomes were contained, with an average of 7 chromosomes. More than half of the cells examined contained human chromosome 14.

〔The invention's effect〕

The heterohybridoma of the present invention is resistant to 8AG, and when it is fused with human lymphocytes as a parent strain, a triple hybridoma can be obtained with high fusion efficiency and cloning efficiency, and Ig is stably produced after fusion. It is a very useful thing that can be done.

[Brief description of the drawings]

FIG. 1 shows the heterohybridoma II in Example 1.
FIG. 3 is a drawing showing changes in the number of cells of a strain in an 8AG-containing medium and a HAT medium. FIG. 2 shows the results of Southern blotting of the heterohybridoma II strain in Test Example 1. In FIG. 2, each lane is as follows. Lane 1: EcoR I treatment for mouse myeloma cells NS-1 Lane 2: EcoR I treatment for heterohybridoma II strain Lane 3: EcoR I treatment for triple hybridoma (C8) 11B-F6 Lane 4: Mouse myeloma cells Hind III treatment for NS-1 Lane 5: Hind III for heterohybridoma II strain
Treatment Lane 6: Hind III treatment for triple hybridoma (C8) 11B-F6 Lane 7: BamHI treatment for mouse myeloma cells NS-1 Lane 8: BamHI treatment for heterohybridoma II strain Lane 9: Triple hybridoma ( C8) BamHI treatment for 11B-F6

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-17688 (JP, A) Fermentation and Industry, 44 [6] (1986) pp. 602-614 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 5/10 C12N 15/02 C12P 21/08 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] (1) Human fetal spleen cells and mouse myeloma cells NS-
An 8-azaguanine-resistant human-mouse heterohybridoma obtained by cell fusion with No.1 and having 66 to 70 chromosomes per cell. 2. The method of claim 1, wherein the human-mouse heterohybridoma comprises 11
The human of claim 1, which is E-5 (FERM P-11094).
Mouse heterohybridomas.