JPH02283278A - Production of antibody-productive cell and production of antibody therefrom - Google Patents

Abstract

PURPOSE:To obtain the title cells capable of high-level secretion of human-type monoclonal antibody useful for therapies or diagnoses in a clinical way by fusion of the tumor cells of mice etc., with the nuclei etc., obtained from human- type monoclonal antibody-productive cells followed by high-density culture and high-level proliferation. CONSTITUTION:Nuclei and/or micronuclei are separated from human-type monoclonal antibody-productive cells (I) prepared by fusion of the human lymphoma cells with the lymph node cells etc., of human pulmonary adenocarcinoma patients, and mixed with mouse or rat tumor cells followed by addition of e.g. polyethylene glycol and then carrying out fusion of the nuclei and/or micronuclei with the mouse or rat tumor cells. The resultant cells are put to culture in a HAT medium to obtain hybridoma. Thence, the resulting culture solution is added to a 96-well microtiter plate followed by making a culture, and cell colonies being in production of human-type anti-human pulmonary adenocarcinoma monoclonal antibody in the resultant culture solution are selected, thus obtaining the objective human-type monoclonal antibody- productive cells (II).

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for producing cells containing nuclei and/or micronuclei derived from human monoclonal antibody-producing cells in mouse or rat tumor cells, and This invention relates to a method for producing human monoclonal antibodies by culturing cells in vlvo or 1n vltro. The human monoclonal antibodies produced by the present invention can be widely applied clinically to therapy and diagnosis.

[Prior Art] Conventional methods for producing monoclonal antibodies include G, which produces monoclonal antibodies using hybridomas produced by fusing lymphocytes of an immunized body with tumor cells;
The method of Kohler and C1M i 1 S T e i n (Nature, vol. 256, p. 495, 1975) is known.

Furthermore, in recent years, using genetic recombination technology, various volatile microorganisms,
It has also been widely attempted to produce monoclonal antibodies by incorporating the genetic information of a desired monoclonal antibody into yeast or animal cells and culturing these cells (for example, J, V.

Brunt, Biotechnology vol. 3, 1
91 pages, 1985).

[Production of monoclonal antibodies by culturing E co-mouse/mouse hybridomas to be solved by the invention is a 1n vivo method using mouse ascites fluid.
In the culture method, approximately 2 to 10 ml of ascites containing 0.5 to 10 mg of monoclonal antibodies per 11 days is usually obtained. However, human
Human hybridomas (hybridomas created from human peripheral lymph cells infected with certain diseases and human tumor cells), human-mouse hybridomas (human peripheral lymph cells infected with certain diseases and mouse tumor cells) human monoclonal antibodies (hybridomas produced from rat tumor cells and human peripheral lymph cells infected with certain diseases) or human-rat hybridomas (hybridomas produced from rat tumor cells and human peripheral lymph cells infected with certain diseases). Monoclonal antibodies derived from peripheral lymphocytes of infected humans) Production of human monoclonal antibodies by cells that produce human monoclonal antibodies cannot be performed in the 1 in vivo culture method because these cells cannot proliferate in mouse or rat ascites. do not have. On the other hand, in the 1nvltro method of culturing the cells, 0.5 to 10μ
Only about 1.5 g of human monoclonal antibodies can be obtained. At present, the production of human monoclonal antibodies is limited to this I
There is no suitable method other than the vltro culture method, and attempts have been made to increase the density of cells, increase the proliferation rate, and search for antibody secreted factors by searching for culture medium composition and improving culture equipment, but these attempts have not been successful. None of these are easy.

An object of the present invention is to provide a method for producing antibody-producing cells that efficiently produce the desired human monoclonal antibody, and a method for efficiently producing the desired human monoclonal antibody.

[Means for Solving the Problems] The present inventors have intensively studied the above problems, and have found that
In order to solve the problems caused by conventional methods, we invented the following method. That is, the present invention provides a human monoclonal antibody producing cell (II) characterized in that the nucleus and/or micronuclear body obtained from the human monoclonal antibody producing cell (1) is fused with mouse or rat tumor cells. and the human monoclonal antibody-producing cells (II) produced by the production method in vlvo or in vitro.
This is a method for producing a human monoclonal antibody, which is characterized by culturing the antibody in a culture medium and collecting the human monoclonal antibody.

In the present invention, human monoclonal antibody-producing cells (1) from which nuclei and/or micronuclear bodies are obtained can be produced by the method of G. Kohler and C1M1lstein, which is known per se. Human monoclonal antibody-producing cells (such as human-human hybridomas, human-mouse hybridomas, human-rat hybridomas, etc., but are limited to any cells that produce human monoclonal antibodies) isn't it.

The method for separating the nucleus of the human monoclonal antibody-producing cells (1) used in the present invention is not particularly limited, but for example, the cells are centrifuged using a Ficoll concentration gradient containing cytochalasin B to enucleate the cells. M, H to separate the cytoplast and nuclear body.

The method of W i g l e r et al. (Biochem.

Biophy, Cell, Res, Vol. 71 = p. 480, 1972).

Also, Co11oidai 5l instead of Ficoll
The method of Bossart et al. using a concentration gradient of lca (Exp, Ce11.Res, Vol. 96, p. 2, 360, 1
975) can also be used.

Furthermore, in order to separate the micronuclear bodies of the human monoclonal antibody producing cells (I), a method of treating the cells with colchicine, etc. can be used.

There are no particular limitations on the method of fusing the nucleus and/or micronuclear bodies obtained from human monoclonal antibody producing cells (I) with tumor cells, but for example, T may be fused using polyethylene glycol.

It can be carried out according to the method of Sekiguti et al. (32nd Japanese Cell Biology Conference: Abstracts, 1979).

In addition, the method of F. H. Ruddle et al. (Proc, Nat 1) using HVJ (Sendai virus)
．． Acad, Sci.

USA, Vol. 74, pp. 319-323, 1977), electrofusion or electroporation methods, and even calcium phosphate methods can also be used.

The human monoclonal antibody-producing cells (n) thus produced are incubated in vitro or in vitro.

By culturing the target human monoclonal antibody, it is possible to efficiently obtain the desired human monoclonal antibody. The culture method is not particularly limited, and may be carried out according to a conventional hybridoma culture method. For example, in the case of in vitro culture, human monoclonal antibody-producing cells (I) may be transplanted into the peritoneal cavity of a mouse, rat, etc., and the purified ascites fluid may be purified to obtain the desired human monoclonal antibody. In in vitro culture, human monoclonal antibody-producing cells (n) are cultured in an appropriate culture medium with the addition of antibody secretion-promoting factors as necessary, and the cells and culture medium are purified to achieve the desired target. Antibodies are obtained.

[Effect of the invention] According to the present invention, human monoclonal antibody producing cells (n
)'s high-density culture, high proliferation, and high antibody secretion in vitro, and furthermore, in vitro proliferation in ascites of mice, rats, etc. is possible, and human monoclonal antibodies can be effectively produced. I can do it.

[Examples] Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited only to these Examples.

(A) Preparation of human monoclonal antibody-producing cells (I) As human monoclonal antibody-producing cells (1), lymph node cells of human lung adenocarcinoma (hereinafter abbreviated as HLA) patients and human lymphoma cells were used. , Koler and C. Mi.
Anti-HLA antibody (IgM) producing cells (I) fused according to the method of Istein et al. were used. For culturing anti-HLA antibody producing cells (I), 15% fetal calf serum (FC5
A DMEM medium containing (abbreviated as) was used.

(B) Preparation of mouse tumor cells Mouse tumor cells include 8
- SP210-Ag14 (as an azaguanine-resistant strain)
(hereinafter abbreviated as S P 210) was used. The cells were cultured in DME containing 15% FCS until the day of cell fusion.
M medium was used.

(C) Isolation of nuclei of anti-HLA antibody producing cells (1) First, cytochalasin B at a final concentration of 10 μg/ml and a final concentration of 0
．． Add 2 ml (25%), 2 mf (17%), 0.5 Ficoll-DMEM (pH 7,4) with a concentration gradient of 12.5-25% containing 5% DMSO to a sterile centrifuge tube.
m1 (16%), 0.5ml (15%), 2m1 (1
2.5%) were prepared in stages with varying concentrations.

The centrifuge tube in which the Ficoll concentration gradient was created must be at least 6
E)H was equilibrated by leaving it in a CO2 incubator for more than an hour.

Anti-HLA antibody-producing cells (I) were prepared by suspending them in DMEM medium and aseptically centrifuging them at 1100 Orp for 5 minutes.

The cells were suspended in a volume of 0.5 to 3.0 x 107 cells per 12.5% Ficoll-DMEM medium and layered on a Ficoll concentration gradient. Further, a serum-free DMEM medium containing cytochalasin B was gently layered on top of the cell layer, and the centrifuge tube was filled to about 3 mm below the top end. At this time, 0.02596) Liban Blue was added to the cell layer and the 16% Ficoll layer to serve as a marker for the layer position.

The centrifuge tube was placed in the rotor, coated with sterile grease, and capped. The centrifugation conditions were 30 degrees and 20,000 to 30,000 rotations for 1 to 2 hours. After centrifugation, the cell layer contained many cell debris and fragments, with 12.5-16% of the layer containing complete cells and cytoplasts, and 17-25% of the layer mainly containing nuclei.

Intact cells and cell fragments were collected at the bottom as a pellet. Fractions were collected without destroying the Ficoll layer at each concentration. Dilute Ficoll solution with DMEM to 2.50
After centrifugation for 5 min at 0 rpm, the cells were washed twice with DME).

(D) Anti-1 (fusion of nuclei of LA antibody-producing cells (I) and mouse tumor cells) The mouse tumor cells prepared in sections (B) and (C) above and the nuclei of anti-HLA antibody-producing cells (I) were After mixing at a ratio of 1:1, the cell pellet was collected by centrifugation (1,000 rpm, 10 minutes).The centrifuge tube was tapped gently to spread the cell pellet on the wall. It was 5
0% PEG (polyethylene glycol 4 manufactured by MERK)
．． 000) in DMEM solution containing 10% DMSO and 5 μg/ml polyarginine (molecular m60,000).
ml was dropped little by little while rotating the centrifuge tube. After mixing by slowly rotating the centrifuge tube for 1 minute,
DMEM, which had been warmed to 37° C., was added 10 times while rotating the centrifuge tube at a rate of 1/2 ml. Next, 2 ml of FCS was slowly added and centrifuged at 11,000 rpm for 10 minutes. Cell pellets in 15% FCS and 1X10 M Hyboxanthin, 4X10 M Aminopterin, 1.6X10
Centrifugal washing was performed twice (1,000 rpm, 10 minutes) with DMEM (hereinafter abbreviated as HAT medium) containing M-thymidine. 200 μl of this culture solution was dispensed into a 96-well microtiter plate (Falcon #3042) at I×10 5 cells/well. The HAT medium was replaced with 100 μl/well every third day. 3
After a week, 1×10 M hypoxanthine, 1.6
DMEM containing X10 M thymidine and 15% FC3
(hereinafter abbreviated as HT medium) was used for medium exchange.

(E) Selection of hybridomas 100 days after cell colonies were observed in the 96-well microtiter plate, solid-phase enzyme immunoassay was performed to examine the presence of anti-HLA antibodies in the culture supernatant.

HLA cells were placed in a 96-well microtiter plate (flat bottom) (Falcon) in DME containing 15% FCS.
Cultured in M medium. Dispense 100 μl/well of PBS solution containing 0.06% glutaraldehyde and incubate for 15 minutes at room temperature.
Cells were fixed for 1 minute. Remove remaining solution in the wells and add phosphate buffered saline (0.85% NaC) to the wells.
1a 0.01% phosphate buffer, pH 7, PB below 27
(abbreviated as S) with 0.04% tween-2
After washing three times with a solution containing 0 (hereinafter abbreviated as PBS-T), 300 μl of a PBS-T solution containing 3.0% bovine serum albumin (hereinafter abbreviated as BSA) was added to each well. Blocking treatment was performed at 37°C for 1.0 hour. Remove remaining solution from wells and incubate with PBS-T for 3
After washing twice, add 1 liter of hybridoma culture supernatant to each well.
00 μl was dispensed and allowed to stand at 37° C. for 1.5 hours. After washing these wells three times with PBS-T solution, 0.
A solution of peroxidase-labeled rabbit anti-mouse IgM antibody (manufactured by Tabo) diluted 4.000 times with a PBS-T solution containing 1% BSA was dispensed at 50 μm/well and allowed to stand at 37° C. for 1.5 hours. After washing three times with PBS-T solution, a substrate solution (1.2% 2.2-7 Ginogy (3-ethylbenzthiazoline sulfate)-diammonium salt (ABT
100 μl of 0.1 M citrate buffer (pH 5.1) containing S) and 0.01% hydrogen peroxide (H2O2) were added to each well. Leave it at room temperature for 30 minutes, and then
The enzyme reaction was stopped by adding 100 μm of M oxalic acid solution. Absorbance at 415 nm was measured, and it was found that human monoclonal antibody producing cells (n) producing anti-HLA antibodies were present in the wells in which enzyme activity was observed.

As described above, monoclonal antibody producing cells (n) with a strong antibody titer were obtained.

(F) Preparation of Condesigination Medium 10ml of refrigerated syringe fitted with a 26 gauge needle.
The 4M sucrose solution was blotted. A Ba1b/c mouse (male) was subjected to vertebrae dislocation, and the above solution was injected intraperitoneally in a sterile manner.

Within 5 minutes after injection, the intraperitoneal solution was collected using a water-cooled syringe with an 18-gauge needle attached to the left flank. Pour the above recovered solution into an ice-cooled centrifuge tube and incubate at 11,000
Centrifuged at rp for 5 minutes.

After centrifugation, discard the supernatant and add 15% FCS-D to the cell pellet.
MEM was added, stirred, and put into a pusher.

The cells were cultured overnight at 37°C, 5% carbon dioxide concentration, and 95% humidity. The culture supernatant was collected, filtered through a 0.22 μm membrane filter, and used as a conditioning medium.

(G) Cloning Anti-HLA antibody producing cells (II) prepared in section (E) above
were made into a single clone using the limiting dilution method. 1ml of the conditioning medium prepared in section CF) above.
20 ml of HAT medium was prepared. The anti-HLA antibody-producing cells (■) to be cloned were prepared in the above culture medium so that one cell per well was dispensed into a 96-well microtiter plate (Falcon #3042) at 200 μm/well. For wells in which cell colonies are observed after 100 days of culture, anti-HLA antibody-producing cells (I) were selected according to the standard method of solid-phase enzyme immunotherapy described in (E) above, and cloning was repeated again. A unique anti-HLA antibody producing cell (n) was established.

(H) In vltro of anti-HLA monoclonal antibody
Production of 1 mg/ml insulin and 7 mg/ml! ) The above CG was added to a DMEM medium containing transferrin, 4mM ethanolamine, and 0.5μM sodium selenite.
The anti-HLA antibody producing strain established in section ) was suspended at 3 x 105 cells/ml and cultured by perfusion using Shimabara 5HC-1.

(1) Measurement of the amount of monoclonal antibody in the culture medium (a) Immobilization of anti-mouse IgM antibody 0.1M sodium carbonate buffer (pH 9,6 200 μl of a solution of 3 μg/ml mouse-derived anti-mouse IgM antibody dissolved in
-Incubated at night. Next, the solution in each well was removed and washed three times with PBS-T solution, followed by 0.1% or less B
300 μl of a PBS-T solution in which SA was dissolved was added to each well, subjected to blocking treatment at 4° C., and stored as is.

(b) Quantification of monoclonal antibodies in samples After returning the microtiter plate prepared by the method described in (a) to room temperature and washing with PBS-T solution, 20 μl of a standard sample containing a mouse IgM antibody was added to each well. added. Next, an anti-mouse IgM antibody labeled with horseradish peroxidase (manufactured by TOYOBO, hereinafter abbreviated as HRP) was diluted with a PBS-T solution, and 200 μl of the antibody was added to each well. After incubating at room temperature for 3 hours, the solution was removed and the plate was washed three times with PBS-T solution. Additionally, 1.2% 2,2-azinody(3-ethylbenzthiazoline sulfate)-diammonium salt (ABTS) and 0
．． 200 μl of a substrate solution consisting of 0.1 M citrate buffer (pH 4.1) containing 0.1% hydrogen peroxide (H2O2) was added to each well, and after enzymatic reaction for 30 minutes at room temperature, 200 mM oxalic acid solution was added. The enzymatic reaction was stopped by adding 100 μm. For each well of the above microtiter plate, wavelength 415nm% control wavelength 492nrn
Automatic microtiter plate reader M
It was measured with PR-A4 (manufactured by Tosoh Corporation, trade name). A calibration curve was created from the relationship between concentration and absorption intensity in this standard sample.

A similar measurement using the culture solution obtained in section (H) above on the 5th day of culture revealed that human anti-HLA monoclonal antibodies were produced at 5 μg/ml.

(J) Chromosome analysis Chromosome analysis of the anti-HLA antibody producing cells (n) established in section (G) above was performed. As a result, it was confirmed that the cells contained human chromosomes.

Claims (3)

[Claims] (1) A method for producing human monoclonal antibody producing cells (II) characterized by fusing the nucleus and/or micronuclear bodies obtained from human monoclonal antibody producing cells (I) with mouse or rat tumor cells. . (2) The method according to claim 1, wherein the human monoclonal antibody-producing cell (I) is a human-human hybridoma, a human-mouse hybridoma, or a human-rat hybridoma. (3) Human monoclonal antibody producing cells (II) produced by the method described in claim 1 or 2,
A method for producing a human monoclonal antibody, which comprises culturing in vivo or in vitro and collecting the human monoclonal antibody.