JPH03259080A - Composition for proliferation of lymphocyte-based cell strain, proliferation method and production of physiologically active substance - Google Patents

Abstract

PURPOSE:To obtain a composition for proliferation of lymphocyte-based cell strains capable of proliferating even started from a single cell by blending a fibroblast cell growth factor (FGF) protein. CONSTITUTION:The objective composition for proliferating lymphocyte-based cellular strains by blending the above-mentioned FGF protein and the method for culturing and proliferating the lymphocyte-based cellular strains in a culture medium is presented. Various kinds of lymphocyte-based cellular strains can be cultured even after inoculation of few cells or even in serum-free culture medium. For example, in the case of a lymphocyte based cellular strain capable of producing physiologically active substance, such as monoclonal antibody, proliferation of cell is promote by addition of FGF protein. Production amount of the physiologically active substance such as antibody is also increased by the promotion of cell proliferation. Consequently, when the production of physiologically active substance such as antibody is carried out using the lymphocyte based cell strain, the physiologically active substance can be efficiently produced and collected by using the objective FGF protein-added serum-free medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a composition for propagating lymphoid cell lines,
This invention relates to a method for proliferating lymphoid cell lines and a method for producing physiologically active substances.

Research on the efficient production of useful physiologically active substances such as interferon, interleukin, and other lycahokines and monoclonal antibodies using lymphoid cell lines is actively progressing from various aspects. It is being On the other hand, fibroblast growth factor (FGF) is Gospodarow
Ioz is a cell growth factor discovered in the bovine pituitary gland (Nature, vol. 249, p. 123, 1974), and it is a cell growth factor that has been found in fibroblasts (Journal of Cell Biology, vol. 66, p. 451, 1975). , Adrenal Cell Yl (Endocrystalline 70G, Vol. 97, p. 1.20 1
975), myoblast cells (Journal of Cell Biology, Vol. 70, p. 395, 1976), chondrocytes (Journal of Cellular Physiology-193)
Vol. 117, 1978), smooth muscle cells (Journal of Cellular Physiology, Vol. 91, p. 377, 1977), vascular endothelial cells (Proceedings of the National Academy of Sciences USA)
, Vol. 73, p. 4120, 1976), it is a cell growth factor that exhibits a proliferation-promoting effect on most cells derived from mesoderm. However, it is known that FGF does not exhibit a cell proliferation-promoting effect on epithelial cells (Cell Growth Factor 1, edited by the Japanese Society of Tissue Culture, Shokusho Shoten, p. 32, 1984).
), and FGF is present in LMW-BCGF (low molecular weight B cell growth factor)-dependent human B lymphocytes.
It has been reported that in the absence of GF, it does not exhibit the effect of promoting cell proliferation (Cellular Immunology, Vol. 122, 42
4 pages 1989). On the other hand, it has not been confirmed that FGF exhibits a cell proliferation-promoting effect on general lincocytic cell lines. Furthermore, it was completely unknown that physiologically active substances could be efficiently produced by culturing in a medium supplemented with FGF. Conventionally, in the culture of lymphoid cells, a medium to which about 10% serum has been added, as in the case of other animal cells, has been mainly used, and in particular, a medium containing tallow serum (Fe2) has been used.

Problems to be Solved by the Invention However, various problems have been pointed out when using a serum-supplemented medium when culturing animal cells to produce useful substances. Specifically, there are lot-to-lot variations in serum, the culture medium is expensive, there is a risk of contamination with mycoplasma and viruses, and there is concern that purification may be complicated and adversely affect product quality.

Therefore, it is advantageous to use a serum-free medium; however, when a serum-free medium is used, cell proliferation is generally poorer than in a serum-supplemented medium, and therefore it is necessary to increase the seeding density during cell transplantation. There are many. For example, serum-free culture of human-human hybridomas producing human monoclonal antibodies required a seeding density of approximately 1X10' cells/-.

When mass culturing is carried out, there is a complication that the cells must be passaged many times, and there is also a high risk of bacterial contamination due to the passage operation. On the other hand, when cloning cells using the limiting dilution culture method, since cells are grown from a single cell, it is essential to add serum or a serum substitute to the medium. An example of a serum substitute is serum-derived growth factor fraction (GFS) [2nd Next Generation Industrial Infrastructure Technology Symposium - Biotechnology Preliminary Lectures, p. 161, 1984].
Since FS also contains a wide variety of components, it is necessary to use a serum-free medium depending on the purpose, but animal cell cloning in a serum-free medium has not yet been achieved.

In order to solve these problems, the present invention aims to provide a serum-free medium that can grow lymphoid cell lines even from a single cell.

Steps to Solve the Problem As a result of extensive research into serum-free media that promote the proliferation of lymphoid cell lines from low seeding densities, the present inventors found that F.
It was discovered that a GF-added medium was suitable for this purpose, and as a result of further research based on this, the present invention was completed.

The present invention provides (1) a composition for proliferation of lymphoid cell lines containing fibroblast growth factor (FGF) protein; (2) FGF;
A method for propagating a lymphoid cell line, which comprises culturing a lymphoid cell line in a lymphoid cell line proliferation medium containing a GF protein, and (3) a lymphocyte containing a FGF protein. This is a method for producing a physiologically active substance, which is characterized by culturing a lymphoid cell line that produces a physiologically active substance in a culture medium for cell line growth, and allowing the physiologically active substance to be produced and accumulated in the culture.

In the present invention, the FGF protein may be any polypeptide or protein that has FGF activity (eg, mammals (eg, humans)).

Natural type F derived from monkeys, pigs, cows, sheep, horses, etc.
Examples of GF proteins include those extracted from various organs whose existence has already been clarified, such as the brain and pituitary gland, but these may also be purified products;
It may also be a crude product such as a tissue extract (eg, bovine pituitary gland extract) containing F protein.

As the FGF protein, commercially available FGF protein may be used, and specific sources include, for example, Wako Pure Chemical Industries, Ltd., Seikagaku Kogyo Co., Ltd., Funakoshi Pharmaceutical Co., Ltd., and Takara Shuzo Co., Ltd. , Toyobo Co., Ltd., SigmaChemic
Cosmo Bio Co., Ltd., Gibco Oriental Co., Ltd., Pellinger Mannheim Yamanouchi Co., Ltd., etc. can be mentioned.

FGF proteins include bFGF protein, which has a basic isoelectric point, and aFGF protein, which has an acidic isoelectric point, and according to the present invention, either type of FGF protein can be applied.

Furthermore, FGF protein obtained by recombinant DNA technology (PCT International Publication No. W○/871
Publication No. 01728; Huebs Letters.

Volume 213, page 189, 1987; European Patent Application Publication No. 237.966 (Japanese Unexamined Patent Publication No. 63-2262)
Publication No. 87)) or FGF Mutin (European Patent Application Publication No. 281,822 (Japanese Unexamined Patent Publication No. 2-193)
Biochemical and Biophysical Research Communications, Volume 151, No. 7
Page 01 1988: European Patent Application Publication No. 326
, No. 907) may also be used.

The above-mentioned FGF mutin is originally a mutation of the amino acid sequence of the original peptide or protein, and therefore, the mutation includes the addition of amino acids.

Examples include deletion of a constituent amino acid and substitution with another amino acid.

Examples of the amino acid addition include those in which at least one amino acid is added.

The deletion of the constituent amino acids includes at least one FG
Examples include those lacking F-constituent amino acids.

As for the substitution with other amino acids, at least one F
Examples include those in which a GF-constituting amino acid is substituted with another amino acid.

The at least one amino acid in a mutin in which at least one amino acid is added to FGF does not include methionine, which is based on the initiation futon used when expressing the peptide, or a signal peptide.

The number of added amino acids is at least one, but any number may be used as long as the characteristics of FGF are not lost.

More preferably, a part or all of the amino acid sequence of a protein that has been recognized to have homology with FGF and exhibits similar activity is included.

The number of missing constituent amino acids in a mutin in which at least one FGF constituent amino acid of FGF is missing may be any number as long as the characteristics of FGF are not lost.

Examples of the deficient constituent amino acids include h) bFG;
Amino-terminal lO residue of F: Met-Pro-Ala-
Leu-Pro-Glu-Asp-Gly-G
1y-5er.

14 amino-terminal residues of human bFGF: Met-Pro
-Ala-Leu-Pro-Glu-Asp4 G Iy -G 1y-S er -G 1y-A l
a - P he - P ro.

Examples include 41 amino-terminal residues of human bFGF and 61 carboxyl-terminal residues of human bFGF. Furthermore, as an FGF mutin, the 7 peptides on the carboxyl terminal side of the original peptide or protein of bFGF are added.
Examples include those lacking 5 to 46 constituent amino acids.

At least one FGF-constituent amino acid of FGF is substituted with another amino acid The number of at least one FGF-constituent amino acid before substitution in an FGF mutin may be any number as long as the characteristics of FGF are not lost.

Examples of the constituent amino acids before substitution include cysteine and cystine, with cysteine being particularly preferred. Constituent amino acids other than cysteine before substitution include aspartic acid, arginine, glycine, serine, and valine.

When the constituent amino acid before substitution is cysteine, the substituted amino acid is preferably, for example, a neutral amino acid. Specific examples of the neutral amino acid include glycine and van.

Alanine, leucine, inleucine, tyrosine phenylalanine, histidine, tryptophan.

Examples include serine, threonine, and methionine. Particularly preferred are serine and threonine.

When the constituent amino acid before substitution is other than cysteine, the substituted amino acid may be, for example, in terms of hydrophilicity, hydrophobicity, or charge of the amino acid.
Choose an amino acid that has properties different from those of the amino acid before being substituted. Specifically, when the amino acid before substitution is aspartic acid, examples of the amino acid after substitution include asparagine, threonine, valine, phenylalanine, and arginine, with asparagine and alkynine being particularly preferred.

When the amino acid before substitution is arginine, examples of the amino acid after substitution include glutamine, threonine, leucine, phenylalanine, and aspartic acid, with glutamine being particularly preferred.

When the constituent amino acid before substitution is glycine, examples of the amino acid after substitution include threonine, leucine, phenylalanine, serine, glutamic acid, and arginine, with threonine being particularly preferred.

If the constituent amino acid before substitution is serine,
Examples of the amino acid after substitution include methionine, alanine, leucine, cysteine, glutamine, arginine, and aspartic acid, with methionine being particularly preferred.

If the constituent amino acid before substitution is valine,
The amino acids that have been replaced are serine, leucine, proline, glycine, and lysine.

Examples include asparachynic acid, and serine is particularly preferred.

Preferred amino acids after substitution are asparagine, glutamine, alkinine, threonine, methionine, serine, and leucine.

The most preferred substituted mutin is one in which the constituent amino acid cysteine is replaced with serine.

In the above substitutions, two or more substitutions may be performed simultaneously. In particular, it is preferred that two or three constituent amino acids are substituted.

The FGF mutin may be a combination of two or three of the above additions, deletions, and substitutions.

The FGF mutin is preferably a mutin in which at least one human basic FGF constituent amino acid is substituted with another amino acid.

The amount of FGF protein added to the medium is approximately 0.1 ng/a.
J~10μg/d, preferably about 10ng/d~2μ
g/d. Further, FGF protein can be added alone or together with hevarin.

When FGF protein is used in combination with heparin, the amount of heparin added is about 1 to 50 μg/min, preferably about 2 to 2 μg/min.
0 μg/rnl is used.

As the medium to which the FGF protein is added, a commonly used serum-containing medium, serum substitute-containing medium, or serum-free medium can be used. The serum-containing medium includes a medium supplemented with 10% tallow serum, and the serum-substitute-containing medium includes:
Serum-derived growth-promoting factor fraction (GFS) [2nd Next Generation Industrial Infrastructure Technology Symposium - Biotechnology Preliminary Lecture Collection 1
An example of this is a medium supplemented with 3 mg/ln1 of [Page 61, 1984]; however, as a medium for producing useful physiologically active substances such as monoclonal antibodies, it is easy to purify;
A serum-free medium is desirable because the medium is inexpensive. Serum-free media include basic synthetic media, insulin, transferrin, ethanolamine, selenite,
A medium supplemented with factors such as polyethylene glycol (preferably insulin, transferrin, ethanolamine, selenite) is used. As the basic synthetic medium, Iscove medium [1scove, N, N, &
Melchers, F., J., ExpWed, I.
Doyu, 923 (197g)), Ham F12 medium [
R,G, Ham, Proc, Natl,
^cad, Sci,, 53. 288 (19
65)), L15 medium [A, Leibovit
z, ^merJ, Hyg,, ヱ8. 173 (196
3)), T medium [JP-A-60-14.5088]
, TL-2 medium (Iscot medium, Ham's F12 medium and L
A 1:1:2 mixture of 1.5 medium) is used, but
Preferably, T medium or TL-2 medium is used.

The composition for propagating lymphoid cell lines containing the FGF protein of the present invention may be in either a solid state or an aqueous solution. used.

The lymphoid cell lines to be cultured by the method of the present invention include established lymphoid cell lines, such as cell lines derived from lymphocytes of mammals such as humans, mice, rats, cows, and hamsters, and lymphoma cell lines. Examples include cell lines derived from leukemia and bone marrow tumors, hybridomas in which one parent cell is a lymphocyte, and lymphocyte cell lines mutated by viruses, etc.

More specifically, mouse metastatic lymphoid cell P3
88 D, (Aier, J., Path, 33.
603 (1957)), human peripheral lymphocytes RPM T-
1788 (J, Nat.

Cancer In5t, 43.1119 (1969)
, human plasmacytoma ARH-77 (Cancer R
es, 38.2508 (197g)), human Burkitt's lymphoma Namalva (lust, J, C
ancer 12.39 & (1973)), Jurk
at(Immunogenetics, 10.247(
1980)).

Human B lymphoblastoid cell line WI-L2 (J,
Exp.

Med, 156.930 (1982)), human acute lymphoblastic leukemia T-cell CCRF-HS
B-2 (Cancer Res.

28, 1.121 (196g)), MOL T-
3(J, Natl, CancerInst, ,
49.891 (1972)), mouse AKR/J-derived T
cell! J 7pa WBW5147. G, 1.4. OUR”
, 1 (J, Natl, Cancer [nst,
, 52.429 (, 1974)), mouse hybrid-7E 235163 (Hybridos+a,
4.47 (19gs)), mouse-human-human heterohybridoma 112 22, 25 (Bioche
m, Biophys, Res, Coaa+un.

, 129.26 (1985)), Human-Human Hybridoma W471-7, 24 (Biochem, B
iophys, Res, Common, , 14
2.805 (1987)), HBW-4, 16, HB
W-6, 20 (Bio/Technology, 7.
374 (19g9)), among which hybridomas producing monoclonal antibodies are preferably used.

Lymphoid cell lines that produce physiologically active substances used in the method for producing physiologically active substances of the present invention include, for example, E235I63, which produces mouse monoclonal antibodies.
, producing human monoclonal antibodies +12-22.2
5. W471-7.24, HBW-4,16, HBW-
Examples include Namalva cells, which produce leukocyte interferon, and Jurkat cells, which produce interleukin-2.

For culturing in the method of the present invention, containers or devices commonly used for culturing are used. For example, multiwell plates,
Culture flask, spinner flask.

A jar fermenter, a Sarani hollow fiber culture device, a culture device using a ceramic matrix, a microcapsule culture method, etc. are appropriately employed.

For the culture of the present invention, conditions suitable for culturing the lymphoid cell line used are employed. - For boat fishing, the culture temperature is approximately 37°C.
It is cultured for several days to three months at around 6.5 to 7.5 degrees Celsius and at a pH of approximately 6.5 to 7.5. For example, the culture medium of the present invention is usually 0.1 to 5X
10' cells/day are seeded, and in the case of a multi-well plate or flask, the pH is about 6.5-7. 5 for about 1 to 20 days. Aeration and agitation culture is performed in jar fermenters and fermenters. In addition, when culturing Cholera in culture vessels, hollow fibers, ceramic matrices, microcapsules, etc., the productivity of physiologically active substances can be improved by replacing the medium batchwise or continuously. In the case of continuous perfusion culture, it may continue for one to several months (about 3 months). It is also ventilated if necessary.

To collect cells from a culture solution, collect the culture solution by directly passing it through a centrifuge or filter. In addition, when a physiologically active substance produced by culturing a lymphoid cell line accumulates in the culture solution, a supernatant is obtained by filtration or centrifugation, and the supernatant is collected from the supernatant. In the case of substances that accumulate within cells, cells obtained by filtration or centrifugation can be treated using physical methods (e.g., ultrasound, French press, Dynomil, etc.) or chemical methods (e.g., guanidine hydrochloride, etc.).
After processing and extracting the product, a supernatant liquid is obtained.

The physiologically active substance can be separated and purified from the supernatant by appropriately combining known separation and purification methods. For example, when the physiologically active substance is a protein or peptide, methods that utilize solubility such as salting out or solvent precipitation, dialysis, ultrafiltration, 1-gel filtration, and 5DS-polyacrylamide gel electrophoresis are mainly used. Methods that utilize differences in molecular weight, methods that utilize differences in charge such as ion exchange chromatography, methods that utilize specific affinity such as affinity chromatography, and methods that utilize differences in hydrophobicity such as reversed phase high performance liquid chromatography. Method 1: A method using a difference in isoelectric points, such as isoelectric focusing, is applied.

The lymphoid cell line grown according to the method of the present invention is
Various lymphoids (e.g. leukocyte interferons, immune interferons, interleukin-2
) and various monoclonal antibodies (e.g., mouse monoclonal antibodies, human monoclonal antibodies, etc.).

Since the method of the present invention allows lymphoid cell lines to be efficiently proliferated, it can be advantageously used as a method for industrially proliferating lymphoid cell lines in large quantities. Furthermore, the method of the present invention allows lymphoid cell lines to be cultured in a serum-free medium even under low seeding conditions, which is advantageous when culturing a large amount of lymphoid cell lines in a serum-free medium. Since lymphoid cell lines can be grown even from scratches, this method is especially advantageous for cloning.

In the method of the present invention, when a lymphoid cell line that produces a physiologically active substance is cultured, the cells are efficiently proliferated, so that the physiologically active substance can be efficiently produced, which is suitable for industrial production. It's advantageous.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

Example 1 Human monoclonal antibody (anti-HBsAg-human MoAb) production against hepatitis B virus surface antigen (Bio/T)
A high-producing antibody strain derived from the following method was inoculated into a 24-well plastic plate at a concentration of I x 103 cells/- in a medium.

As the medium, serum-free medium PEG-85-1 (Appl, Micro
biol, Biotechnol, 27.5ia (
ta88)) was used to dispense 1 volume per hole. b
FGF was purchased from Cosmo Bio Co., Ltd. (C
ollaborative Re5earch Inc.
(manufactured by), and the concentration in the medium was 0.1. ng/-~40
It was added at a concentration of μg/−. When the cells were cultured for one week in a 5% carbon dioxide incubator at 37°C and the number of cells was counted, b
The effect of adding FGF was observed. The number of cells is 0.2X in bFGF-free medium], O' cells/g, and the optimal concentration range (16
C) -1200 ng/d bFGF) in bFGF supplemented medium, approximately 1.2X1. It was O' pieces/storehouse. That is, b
With the addition of FGF, the cell number increased approximately 6 times that without addition.

Example 2 An experiment was conducted in the same manner as in Example 1. Culture was carried out for 2 weeks, and after culture, the number of cells and anti-HBsAg-human Mo
Ab production was measured. In the bFGF-free medium, the cell number was 0.9 x 10' cells/death, and the antibody production amount was 1 μg/-, but in the bFGF-added medium at 600 ng/-,
Cell count: 50 x 10' cells/ml.

Antibody production amount 10.5μg/Wt1. It became. That is, the addition of bFGF increased the number of cells by about 50 times and the amount of antibody produced by about 10 times.

Example 3 An experiment was conducted in the same manner as in Example 1. However, the medium is P
Culture was carried out for 2 weeks using EC, -86-1 medium supplemented with heparin at a concentration of 6 μg/depletion. After culturing,
When the number of cells was counted, it was 0.9 x 10 cells/- in bFGF-free medium (heparin added), and about 40XlO' pieces/
- became. That is, compared to bFGF-free medium, b
In the FGF-added medium, approximately 40-fold promotion of cell proliferation was observed. In addition, from comparison with Examples 1 and 2, by adding heparin to the culture medium in combination with bFGF, bFGF
It has been found that the minimum effective concentration of GF can be reduced.

On the other hand, from comparison with Example 2, it is clear that adding heparin alone to the medium does not have a cell proliferation promoting effect.

Example 4 An experiment was conducted in the same manner as in Example 3. However, instead of bFGF, aFGF (purchased from Cosmo Bio Co., Ltd., Co11aborative Research
Inc.) was added. The number of cells after 2 weeks of culture is
In the aFGF-free medium, the number was 0.9×10′ cells/cell, and in the aFGF-added medium at the optimal 211 degrees range, it was about 35×IO′ cells/cell. The optimal concentration range of aFGF is bFGF
The cell proliferation effect of aFGF is not much different from that of bFGF.
There was no big difference.

Example 5 Mouse metastatic lymphoid cell P388D, human peripheral blood lymphocyte RPMI-1788, human plasmacytoma ARH-
77, human Burkitt's lymphoma Namalva and human B lymphoblastoid cell line WIL2 were each implanted into a 24-well plastic plate at 1×10 3 cells/− in the medium.

The medium used was PEG-86 without bFGF (manufactured by Funakoshi Pharmaceutical Co., Ltd.) and with various concentrations of bFGF.
Using 1 medium, 11 nl was dispensed per well of the plate. When cultured for 8 days in a 5% carbon dioxide gas incubator at 37°C, the proliferation of all cells was promoted by the addition of bFGF. That is, P388D was 1. compared to when no bFGF was added in cells. 3 μg/ln1 bF
Addition of GF increased A to about 4 times, and in RPMI-1788 cells, addition of 1.3 μg/- bFGF increased A to about 16 times.
In RH-77 cells, the increase was approximately 6 times by adding 0.6 μg/ml of bFGF, and in Namalva cells, it was 1.3 μg/ml.
i;! in wr-L2 cells by about 4 times by addition of bF GF. Addition of 0.6 μg/d of bFGF increased the number of cells approximately three times.

Example 6 A high antibody producing strain induced by the anti-HBsAg-human MoAb-producing human-human hybridoma HBW-4,16 was implanted into a 96-well plastic plate in a medium at 0.5 cells per hole. . As a medium, P
EG-86-1 medium (additive-free medium), bFGF (
Purchased from Cosmo Bio Co., Ltd., Gollabora
PEG-86-1 medium supplemented with H μg/d and bovine serum albumin (B
SA) Using PEG-86-1 medium supplemented with 1 μg/-,
1001 L (!) was dispensed per well and cultured for 15 days in a 5% carbon dioxide gas incubator at 37°C. In the additive-free medium, 6 96-well plastic plates, or 5
No cell proliferation was observed in all 76 wells, or b
In the FGF-added plate, cell proliferation was observed in 11 out of 576 wells. However, cells did not proliferate at all on the plate to which BSA was added instead of bFGF.

Therefore, the effect of adding bFGF is not simply the effect of adding a general protein, but is thought to be due to the growth promoting activity of bFGF on lymphoid cell lines.

Effects of the Invention By using a medium supplemented with FGF protein, various lymphoid cell lines can be cultured in a serum-free medium even under low seeding conditions. In the case of lymphoid cell lines that produce physiologically active substances such as monoclonal antibodies, the addition of FGF protein promotes cell proliferation and increases the amount of physiologically active substances such as antibodies produced. When producing physiologically active substances such as antibodies using cell lines, the physiologically active substances such as antibodies can be efficiently produced (produced and collected) by using a serum-free medium supplemented with FGF protein.

Furthermore, by adding FGF protein, it is possible to clone various lymphoid cell lines in a serum-free medium. This means that single cells can be selected in a serum-free medium, which is extremely useful for breeding and improving lymphoid cell lines such as hybridomas.

Claims (7)

[Claims] (1) A composition for propagating a lymphoid cell line containing a fibroblast growth factor (FGF) protein. (2) The composition according to claim 1, which does not contain serum. (3) The composition according to claim 1 or 2, further comprising one or more of insulin, transferrin, ethanolamine, and selenite. (4) A method for propagating a lymphoid cell line, which comprises culturing the lymphocyte cell line in a lymphoid cell line proliferation medium containing fibroblast growth factor (FGF) protein. (5) The method for propagating lymphoid cell lines according to claim 4, wherein the medium for propagating lymphoid cell lines is a serum-free medium. (6) Culture lymphoid cell lines that produce physiologically active substances in a lymphoid cell line growth medium containing fibroblast growth factor (FGF) protein, and add physiologically active substances to the culture. A method for producing a physiologically active substance characterized by producing and accumulating it. (7) The production method according to claim 6, wherein the lymphoid cell line growth medium is a serum-free medium.