DD232509A5 - METHOD FOR THE PRODUCTION OF LYMPHOTOXINE AND LYMPHOTOXIN-MRNA - Google Patents

Abstract

The invention relates to a process for the preparation of lymphotoxin and lymphotoxin mRNA for use in the prophylaxis and for the control of tumor cells. The aim of the invention is to provide a new method by which lymphotoxin and lymphotoxin mRNA can be produced in sufficient quantity and purity. The novel process for producing lymphotoxin or lymphotoxin mRNA is carried out according to the invention in such a way that virus-transformed monkey T lymphocytes are cultivated in a suitable medium in permanent culture and the lymphotoxin or lymphotoxin mRNA is isolated by methods known per se becomes. According to the invention, the viruses used are preferably herpesviruses, but preferably herpesvirus saimiri or herpesvirus ateles, and cells from the genus Saguimus as monkey T lymphocytes.

Description

AP C 08 G / 266 738/8 (64 316/18)

Process for the preparation of lymphotoxin and lymphotoxin-mRIJA Field of application of the invention

The invention relates to a process for the preparation of lymphotoxin and lymphotoxin mRITA. The lymphotoxin and lymphotoxin mRUA prepared according to the invention are used for the prophylaxis and control of tumor cells.

Characteristic of the known technical solutions

It is known from the literature that certain mammalian cells, after appropriate in vitro stimulation, are capable of producing so-called lymphotoxins (see, for example, Evans in Cancer Immunol, Immunother, 1_2, 181-190 (1983) and Granger et al., Biology of the Lymphokines ρρ 141-180, Academic Press 1979). Herein, lymphotoxin does not represent a single protein in vitro in a given species, but rather describes a number of subtypes that differ in molecular weight, charge and stability (see, for example, Granger et al., Biology of the Lymphokines, pp. 141-180, Academic Press 1979) · In the human system, for example, four classes of lymphotoxins are distinguished, their molecular weights ranging from 10,000 to 20,000 for JTiLT, 35,000 to 50,000 for BLt, 70,000 to 90,000 for osLQ? and 200,000 to 600,000 for LT complex lie.

Lymphotoxins have the ability in vitro

a) to prevent the malignant transformation of cells (see Evans et al in Int J. Cancer 2J _-.., 45-49 (1981) and Eansom et al in J. Hatl Cancer Inst · 6 £, 741-744 (.. 1982)),

b) to act cytostatically or even cytolytically on tumor cells (see Evans et al in Inununopharmacology 3_ '347-359 (1981)) and

c) to increase the sensitivity of tumor cells to enhance the cellular immune defense of the organism (see Evans et al., Cell Immunol 63, 1-15 (1981) and Ransom et al., Int.J. Cancer 29, 451- 458 (1982)).

Furthermore, it is known from the literature that lymphotoxin preparations in the animal model can also cause the regression of tumors (see Khan et al., Proc Soc., Ex. Biol. Med. 169 , 291-294 (1982)). Lymphotoxins are therefore of great importance for the prophylaxis and treatment of malignant diseases.

The hitherto known methods for obtaining lymphotoxins are based in most cases on primary cells. eg, leukocyte preparations from human peripheral blood or tonsils (see Williams et al., J. Immunol., 130 , 518-520 (1983)), mouse spleen cells (see Aksamit et al., Infect. 1028-1035 (1982) and Trivers et al., J. Immunol., 117 , 130-135 (1976)) or from abdominal cavities of Syrian hamsters or guinea pigs (see, for example, Evans et al., Int. J. Cancer 2_7_ , 45-49 (1981)). These cells must first be stimulated by incubation with high molecular weight mitogens, such as phytohemagglutinin, for the production of lymphotoxins. So far, only low lymphotoxin activities have been found in the culture supernatants of B lymphocyte cell lines (see Granger et al., J. Immunol., 104 , 1476 (1970) and Amino et al., J. Immunol., 113. _f 1334-1345 (1974)).

Since, according to the methods used hitherto for the production of lymphotoxins, only small amounts and only low purity could be produced, the hitherto used

Lymphotoxins were neither purified to homogeneity nor provided in sufficient quantities for more extensive investigations of the antineoplastic action on the animal model. In addition, no biologically active lymphotoxin mRITA has yet been detected; however, this is the prerequisite for the molecular cloning of the lymphotoxin genes by the methods of genetic engineering,

Object of the invention

The aim of the invention is to provide a method by which lymphotoxin and lymphotoxin mRNA can be produced in sufficient quantity and quantity.

Explanation of the essence of the invention

The invention has for its object to find a suitable starting material which is capable of producing in a suitable culture medium lymphotoxin and lymphotozine mRliA.

According to the invention, it has now been found that cells of the so-called T lymphocyte type of monkeys, in particular of the genus Saguinus, for example the cell lines L-77/5 "A651, A2543, 70ΪΓ2, 1022 and 167O, but preferably the cell lines 1022 and 1670, the by infection with viruses, preferably with herpesviruses such as herpes virus saimiri or herpes virus ateles, transformed in vitro or in vivo and enabled to permanent growth ± n culture, in a suitable culture medium spontaneously, that is, without further induction, significant amounts of LyphotoxLn and produce lymphotoxin mRUA.

Perner was found dai3 · by adding a sogn · stimulator to the culture medium 25 as a literature known tumor promoter such as a taxane derivative, preferably from Tiglian- or daphnane-IYP "z * B _# Mezerein or an ester of phorbol, preferably ^ - Q-tetradecanoylphorbol - ^ - acetate (see Diamon et al · in Advances in Cancer Research Vol 32 ·, ^s nits 1 to 74 _f Academic Press 1980 and Hecker in Garcinogenesis · Vol II, Mechanisms of umor ™ Tromotion and Oocarcinogenesis, pages 11 -48, Raven Press, New York, 1978), in a concentration of 1-1000 ng / ml, the production of lymphotoxin and lymphotoxin-mRIIA is further increased.

Suitable culture media are the usual serum-free and serum-containing culture media, for example Eagle's Minimum Essential Medium or Roswell Park Memorial Institute Medium 1640 (RPMI1640), to each of which fetal calf serum and antibiotics can be added, preferably up to 10% fetal calf serum, penicillin and streptomycin ,

In the production of lymphotoxin proteins, the method of the invention is preferably carried out in such a way that transformed cells after addition of 10 to 100 ng / ml 12-0-tetradecanoylphorbol-13-acetate or mezerein as a stimulator in a serum-containing, under certain conditions also in a serum-free culture medium, eg in RPMI 1640, for 1 to 7 days, but preferably for 3 to 4 days, and then the lymphotoxin thus formed is separated from the culture supernatant by methods known per se, concentrated and purified. However, this method is particularly advantageously carried out in such a way that the cells after initial proliferation in a serum-containing medium in a serum-poor, but preferably serum-free medium, converted.

In the production of lymphotoxin mRNA, the method according to the invention is preferably carried out by transforming cells after addition of 10-100 ng / ml of 12-0-tetra-decanoylphorbol-13-acetate or mezerein as a stimulator in a serum-containing culture medium, e.g. are incubated in RPMI1640 and 10% fetal calf serum for 6-48 hours, preferably 12 to 24 hours, then the cells are separated and, after destruction, the mRNA is isolated according to methods known per se.

In the isolation of the cell culture products thus formed, in the purification of the lymphotoxin thus obtained, the chromatography on pore-controlled glass, which

maize was used, and, after destruction of the cell membranes and removal of cell nuclei, the extraction of lymphotoxin mRNA, for example with phenol proved to be particularly suitable.

The further purification of the lymphotoxin is carried out, for example, by means of HPLC.

The resulting lymphotoxin having a molecular weight of 60,000 + 15%, e.g. in aqueous isotonic solution, for the prophylaxis and for the control of tumor cells.

Compared with the prior art, the method according to the invention has the following advantages:

1. A homogeneous cell population with unlimited viability is used, the multiplication of which can be extended to any scale,

2. The production of lymphotoxin in these cultures takes place spontaneously, that is, no high molecular weight mitogens have to be added,

3. the production of lymphotoxin can be further increased by low-molecular stimulators,

4. the production of lymphotoxin can be done in serum-free medium with high yields; This gives an excellent starting material for further protein-chemical purification and

5. The RNA isolated from the cells immediately shows "detectable lympho-toxin mRNA activity and thus represents an excellent starting material for the further enrichment of this mRNA, which in turn is an essential prerequisite

for the molecular cloning of the mRNA. The cloned monkey lymphotoxin mRNA can in turn be used by known methods as a probe for the isolation of the homologous human lymphotoxin mRNA or the corresponding genes.

-7- Ausfühnuigsbeispiel

The following examples are intended to illustrate the present invention without, however, limiting it:

example 1

Production of Ljrmphotoxin by Herpesvirus-Transfected Monkey T Lymphocyte Cell Lines

The lymphoid cell lines used were kept in Roswell Park Memorial Institute Medium 1640 (RPMI 164O) with added 10 % fetal calf serum, penicillin and streptomycin in stationary culture at 37 ⁰ C in an atmosphere of 95 % air and 5% carbon dioxide and three times a week diluted in fresh medium. In all experiments, dense cultures were diluted 1: 2 to 1: 3 with fresh medium, diluted 1: 3 the next day, and 5 ml each were made up in a culture flask. The culture supernatants were harvested by centrifugation after three days frozen and stored until testing at -20 ⁰ C ·

The following table contains the lymphotoxin activities determined in three different culture supernatants of each cell line:

cell line origin reference Expt. 1 lymphotoxin 3 1 RE / nvl (transforming 1 Expt. 2 expt. Herpesvirus = HV) 25 L-77/5 Saguinus oedipus - lymph nodes A 3 a tumor-bearing animal 15 (HV saimiri) 48 A 651 Saguinus oedipus - in vitro Trans B 30 formation of lymphocytes 90 (HV ateles) 50 A 2543 Saguinus oedipus - in vitro Trans B 56 formation of lymphocytes 56 (HV ateles) 630 70N2 Saguinus oedipus - Thymus A, C, D, E 47 a tumor-bearing animal 580 77 (HV saimiri) 1022 Saguinus oedipus - tumor D 88 480 (HV ateles) 1670 Saguinus nigricollis - spleen A, C, E 83 a tumor-bearing animal (HV saimiri)

References:

A = Fleckenstein et al., Int. J. Cancer 19, 546-554 (1977) B = Abb et al., Cancer Immunol. Immunother. 9, 219-226 (1980) C = Faik et al., Bacteriol. Proc. 38, 191 (1972)

D = Johnson et al., Proc. Natl. Acad. Be. USA 7j [, 6391-6395 (1981)

E = Kaschka-Dierich et al., J. Virol. 44, 295-310 (1982)

Review: Fleckenstein, Biochem. Biophys. Acta 560 , 301-342 (1979)

Moving cultures were used instead of stationary cultures to produce larger numbers of cells. For this purpose, the cells (for example, line 1670) were, for example, in RPMI 1640 medium supplemented with 10% fetal calf serum in 1- or 2-liter Erlenmeyer flasks on rotary shakers (0.5 1 culture per 1 1 flask, 1 1 Culture per 2 L flask, 40 revolutions per minute (37 ° C in normal atmosphere) and diluted three times a week with fresh culture medium in a ratio of 1: 2 to 1: 3. In this way, the production of culture supernatant in the order of 10-100 1 weekly is readily possible.

The production of lymphotoxin in 1670 cells was stable for long periods of time: after 9 months in continuous culture, no significant decrease in lymphotoxin activity was seen in the culture supernatants.

- 10 characterization of lymphotoxin;

a) The following detection method was used for the biological detection of lymphotoxin activity (modified according to Trivers et al., in J. Immunol., 117 , 130-135 and Evans in Cell, Immunol., 63, 1-15 (1981)):

Cell line L929 transformed mouse cells were cultured overnight with 1 microCurie 3H-thymidine / ml culture medium (preferably Eagle's minimum essential medium with 10% fetal calf serum), then trypsinized and suspended in fresh culture medium with 5% fetal calf serum. 0.5 ml of this suspension containing about 30,000 cells were pipetted into culture dishes (diameter 16 mm) and incubated for 3-6 hours. Subsequently, serial dilutions of the sample to be tested were applied and incubated for three days (all incubations at 37 ° C). The radioactivity in the culture supernatants was finally measured in the liquid scintillation counter.

The following figure shows the detection of the lymphotoxin activity of a culture supernatant of 1670 cells. Each dilution was tested in parallel in 4 culture dishes. Only the mean values were reported, the standard deviation was 3-4% for the first three dilutions and 6% for the highest dilution. The culture supernatant used in this experiment was frozen in several hundred vials and carried as reference standard in all lymphotoxin assays; its activity was arbitrarily taken at 100 reference units per milliliter (RE / ml):

3000

2000

S 1000-

1: 216

1 1 = 1296

DILUTION

Culture supernatant from 1670 cells controls (medium)

For comparison, culture supernatants of a series of human T-lymphocyte cell lines (MOLT-4, 1301, JURKAT, Karpas-45, CCRP-CEM and CCRF-HSB-2) were examined in the above-mentioned biological lymphotoxin test. In no case could a significant increase in 3H-thymidine release be demonstrated over untreated controls.

b) Physico-chemical characterization of lymphotoxin activity in culture supernatants of cell line 1670 was performed by the following experiments:

Stability to thermal stress Stability to acidic pH Stability to freezing / thawing Stability to sodium dodecyl sulfate (SDS) Stability to reducing agents (2-mercaptoethanol)

Molecular Weight Determination by High Pressure Liquid Chromatography (HPLC)

The following table shows the results of the stability tests:

treatment 4 ° C Lymphotoxin activity in percent of control 37 ° C cheap 56 ° C - 5 hours 37 ° C 68% 5 cycles freezing / thawing 108% pH 2.0 - 24 hours at 1 % 0.1% SDS - 1st hour at 15%, 0.1 M 2-mercaptoethanol - 1 hour at 95%

The activity is very stable to mercaptoethanol over heating and several cycles of freeze / thaw, and is completely destroyed at pH 2 within 24 hours.

Thermal and pH stability and freeze / thaw experiments were performed with the standard culture supernatant from untreated 1670 cells in the presence of 10%.

fetal calf serum. The other two experiments were carried out on a culture supernatant concentrated and enriched with porose-controlled glass (see Example 4) of 1670 cells treated with Mezerein (20 ng / ml, see Example 4) (protein content 1-2 mg / ml) Incubation with SDS or mercaptoethanol. with complete culture medium (10% calf serum) diluted 50-fold.

The molecular weight of the lymphotoxin from 1670 cells was determined by high pressure liquid chromatography (HPLC - see the following figure):

5 ο Λ 1 ³ "O i \ UJ 2- J \ 1 τ \ S 1- / I I Λ

1 1 1 i I 1 I I Γ 20 30 40

RETENT1ONSZE1T (MINUTES)

200 microliters of a culture supernatant of untreated 1670 cells concentrated over porose-controlled glass were separated by HPLC on a WATERS plant (2 columns 1-125-20 mM sodium phosphate pH 7, 1M NaCl, 0.1% Tween 20 (polyoxyethylene sorbitan). monolaureate, n = 20, MW: about 1200), 25% (v / v) propylene glycol, 0.5 ml per minute).

0.5 ml fractions were collected and tested. As calibration proteins for determining the molecular weight, serum albumin, ovalbumin, trypsinogen and lysozyme were separated in the same system. The analysis showed a single activity peak at a molecular weight of 60,000 + 15%,

c) The growth-inhibiting effect of culture supernatants of cell line 1670 was examined on a series of transformed (tumor) cell lines. In these experiments, the tumor cells (cultured in Eagle ¹ s Minimum Essential Medium with 10% fetal calf serum, penicillin and streptomycin) in culture dishes recognized (50,000 cells per dish 3 cm in diameter) and a few hours later with medium or the standard culture supernatant of Cell line 1670 (final concentration 16 RE / ml, 3 ml / dish). After 3 or 4 days of incubation at 37 ° C., the cell number per dish was determined (in each experiment, two dishes were used in parallel with lymphotoxin or medium). For three cell lines the number of cells was determined daily. The results are shown in the following table or figure:

species Cell line Λ Incubation period (days) Cell count / 3 cm culture dish (10E-5 χ cells / dish) control lymphotoxin human Heia Hep-2 A-549 4 3 4 8.1 0.6 9.3 2.1 10 3.4 monkey GL-V3 Vero 3 3 5.6 4.5 6.3 5.3 mouse L9 29 IT22 B16 4 3 4 8.5 3.1 13 4.0 19 4.9 rabbit RK13 3 4.6 3.5

10 " ⁴ * CELLS PER BOWL

O-

O-

Where-

• Controls O Lymphotoxin

These show that culture supernatants from 1670 cells can inhibit the proliferation of tumor cells of different species. This property is characteristic of lymphotoxins (Evans in Cancer Immunol., Immunother. 12: 181-190 (1983)); it distinguishes this group of proteins, in particular the interferons, which are also capable of inhibiting the growth of tumor cells, but are generally species-specific, with human interferon being very little or not at all active on mouse cells.

The above-mentioned transformed human T-lymphocyte cell lines and the (tumor) cell lines are known from the literature.

Cell line 1670 was deposited on 28 March 1984 under number 1-294 with the Collection national de cultures de microorganismes (C.N.C.M.) Institut Pasteur, Paris "under Rule 28 of the European Patent Convention.

Example 2 "

Increased lymphotoxin production in monkey T lymphocyte cell lines by diterpene compounds

Densified cultures were diluted 1: 2 to 1: 3 with fresh medium, diluted 1: 3 again the next day, and divided into 5 vials of the same stock culture in 3 vials. The stimulators were added (stock solutions 100 micrograms / ml in dimethylsulfoxide) and the cultures were incubated for three days. Subsequently, the cells were removed by centrifugation, the supernatants frozen and stored at -20 ⁰ C until the lymphotoxin test.

The following table shows the effect of two selected diterpene tumor promoters, 12-0-tetradecanoyl-phorbol-IS-acetate (TPA) and Mezer, on lymphotoxin production in selected monkey T-lymphocyte cell lines:

. ' \:

cell line Exper iment Concentration (ng / ml) Lynipho toxin-A control Liveness in Ü TPA supernatant (RE / ml) Mezerein 1670 1 20 83 520 270 2 20 77 210 76 3 100 88 340 370 70N2 1 20 56 310 280 2 20 47 120 240 3 100 50 65 92 4 100 12 130 160 A651 1 20 260 360 370 • 2 100 130 320 270 A2543 1 20 56 140 190 2 100 48 190 180 L77 / 5 1 20 3 36 86 2 100 1 38 35 1022 1   ioo 480 760 930 2 100 630 700 610

Example 3 Production of lymphotoxin in serum-free medium

After proliferation of the cells in serum-containing medium (analogous to Example 2), the cells were transferred to serum-free medium.

The following table shows a comparison of lymphotoxin production in 1670 cells analogously to Example 1 in each case in serum-containing and serum-free medium in the presence of 20 ng Mezerein / ml:

Experiment Serum content of the medium Lymphotoxin activity Protein content Special acti {%) (RU / ml) (Mg / ml) tivity (RE / mg) 1 2 10 10 370 180 5 5 74 36 3 10 270 5 54 4 0 140 0050 2800 5 0 72 0053 1400 6 0 160 0044 3700

Protein determination was performed by the method of Bradford (see Anal.Biochem., 72, 798 (1976).

- 20 Example 4

Concentration and purification of lymphotoxin from 1670 cells by chromatography on columns from pore-controlled

Glass ;

1670 cells were grown in RPMI-1640 medium with 10% fetal calf serum in rotating Erlenmeyer flasks (0.5 liter culture in a 1 liter flask, rotation rate 40, - · revolutions per minute). Cells from well-growing cultures ? ' , Were recovered by centrifugation, washed and resuspended in serum-free RPMI-1640 medium supplemented with 20 ng / ml of mezerein. The suspension was kept stationary in 150 cm plastic culture bottles (200 ml per bottle) for three days, then the culture supernatant was recovered by centrifugation and filtration.

Thus prepared culture supernatants were chromatographed over 10 ml of pore-controlled glass (pore diameter: 350 angstroms, grain size: 120-200 mesh, column diameter: 9 mm, flow rate: 140 ml / hour). Subsequently, the column was treated with 10 mM sodium phosphate buffer

• pH 7.2 / 1.5 M NaCl and finally washed with the same

'v ' i buffer eluted in 50% ethylene glycol (v / v).

The following table gives the results of two such experiments:

t Experiment 1 volume lymphotoxin protein specific activity Sample Run Wash Fraction Eluate Experiment 2 (Ml) (RE / ml)% (Mg / ml) RE / mg Sample eluate 2,800 2,800 10 20 162 100 13 8 96 1 17.300 76 0.044 2.0 3,700 8,650 2,500 17 141 100 8,600 41 0.050 1.6 2,800 5,400

-22- Example 5

Detection of lymphotoxin mRNA activity in RNA preparations from 1670 and 1022 cells

The cells were suspended in 800 ml of cell culture medium containing 10% fetal calf serum at a cell density of about 5 σ 10 cells / ml and added with 20 ng of Mezerein / ml. Twenty hours later, the cells were harvested by centrifugation, in a suitable buffer (e.g. 10mM HCl · HCl pH 7.4, 140mM NaCl, 1.5mM MgCl ₂ ) and dissolved in 10mL of lysis buffer (for example 10mM Iris.HCl pH 8.4, 140mM NaCl, 1mM) , 5 mM MgCIp, 0.5% Nonidet-P suspended 40 (non-ionic detergent, trademarks) · After lOminütiger incubation in an ice bath, the suspension was briefly shaken and the nuclei removed by centrifugation. from the supernatant of the centrifugation (= cytoplasm) was carried on The procedure was known, for example by extraction with phenol (see Aviv et al., in Proc Natl Acad., U.S. 6, 1408-1412 (1977)), which was finally dissolved in water (concentration about 3 to 5 mg / ml) and by methods already known per se in Xenopus laevis - O ocyten translated (see, for example, Colman et al. in Cell .12, 517-526 (1979), Hitchcock et al. in anal. Biochem. 109 , 338-344 (1980) and Oontreras et al, in Anal. Biochem. VQ, 185-187 (1981). The translation products were tested in the biological test for their lymphotoxin activity.

The following table shows the cytotoxic activity found in oocyte supernatants as compared to RNA preparations from MOLT-4 cells, a human T lymphocyte cell line whose culture supernatants have no detectable lymphotoxin activity, and supernatants from untreated oocytes:

preparation

Supernatant radioactivity (cpm / 100 microliter) Oocyte dilution supernatants

12

42

147

514

Controls (uninjected oocytes)

MOLT-4 RNA Expt. 1

Expt. 2

1670 RNA Expt. 1

Expt. 2

Expt. 3

2200 2300 2100 2500 2400 2200 1800 3000 1600 2400 1700 2200 1700 2300 1600 1800 4700 4700 3600 2800 4300 3800 3300 2600 3200 2800 2800

controls

1670 RNA Expt, 1 1670 RNA, Expt. 2 4,400

6,100 6,600

3300

5,000 5,100

1022 RNA Expt. 1 7,000

5000

The table shows that all three RNA preparations from 1670 cells and an RNA preparation from 1022 cells at up to 147-fold dilution caused a significant increase in the released radioactivity, while RNA from MOLT-4 cells has no such activity , These experiments demonstrate that biosynthetic lymphotoxin mRNA is present in RNA preparations from 1670 and 1022 cells.

Claims (13)

-25-claim. A method of producing lymphotoxin or lymphotoxin mRNA, characterized in that virus-transformed monkey T lymphocytes are cultured in a suitable medium in permanent culture and the lymphotoxin or lymphotoxin mRHA is isolated by methods known per se · 2. Method according to item 1, characterized in that herpesviruses, but preferably herpesvirus saimiri or herpesvirus ateles, are used as viruses, 3. The method according to item 1, characterized in that cells from the genus Saguinus are used as monkey T lymphocytes. 4 * Method according to point 3 », characterized in that the lines 1022, 1670, 70N2, L77 / 5, A651 or A 2543 are used as T-lymphocyte cell lines · Method according to item 3, characterized in that the lines 1022 or 1670 are used as T lymphocyte cell lines. 6 * Method according to items 1 to 5 », characterized in that up to 10% fetal calf serum is added to the culture medium. 7. The method according to the items 1 to 6, characterized in that the. Culture medium for production stimulation low molecular weight tumor promoters, preferably diterpenes, in particular of Daphnan, ingelan and Tigliantyp, or Indolalkaloide be added · 8 · Method according to points 1 to 7, characterized in that for the stimulation of the production of lymphotoxin during incubation as a tumor promoter an ester of phorbol or Mezerein, in a concentration of 1-1000 ng / ml, but preferably of 10-100 ng / ml, is added · * Method according to points 1 to 7, characterized in that for the stimulation of the production of lymphotoxin mKNA during the incubation as tumor promoter an ester of phorbol or mezerein, in a concentration of 1-1000 ng / ml, but preferably of 10 -100 ng / ml, is added 10. Method according to points 1 to 8, characterized in that after proliferation of the cells in serum-containing medium, the cells are transferred to an aerum-free medium. 11. Method according to points 1 to 8 and 10, characterized in that the lymphotoxin is separated from the culture supernatants after 1 to 7 days, but preferably after 2 to 4 days. Method according to points 1 to 8, 10 and 11, characterized in that the lymphotoxin is enriched via a column of pore-controlled glass. Method according to items 1 to 7 and 9, characterized in that the cells are incubated in a culture medium, the fb'talea calf serum, and the lymphotoxin-mEHA is incubated from the cells after 3 to 48 hours, but preferably after 6 until 24 ^s , is isolated · Hienu 3 soaps ~ Zt \ c