NO843431L - PROCEDURE FOR THE PREPARATION OF Lymphocytoxin OR Lymphocytoxin mRNA - Google Patents

Abstract

Method for producing lymphotoxin and lymphotoxin mRNA by incubating transformed T lymphocytes in continuous culture and subsequently separating the cell products thus formed.

Description

It is known from the literature that certain cells from mammals, after appropriate stimulation, are able to produce so-called lymphotoxins in vitro (see for example Evans in Cancer Immunol. Immunother. 12, 181-190 (1983) and Granger et al. in Biology of

the Lymphokines pp. 141-180, Academic Press 1979). In these cases, lymphotoxin from a specific species does not constitute a uniform protein in vitro, rather a number of subtypes are described that differ from each other with regard to molecular weight, charge and stability (see for example Granger et al. i. Biology of the Lymphokines p. 141 -180, Academic Press 1979). In the human system, for example, a distinction is made between four classes of lymphotoxins, where the molecular weights lie in the range from 10,000 to 20,000 for yLT, 35,000 to 50,000 for ØLT, 70,000 to 90,000 for aLT

and 200,000 to 600,000 for LT complex.

In vitro, lymphotoxins have the ability to

a) prevent the malignant transformation of cells (see Evans et al., Int. J. Cancer 21_, 45-49 (1981) and Ransom et al., J. Nat. Cancer Inst. 69, 741-744 (1982)) , b) act cytostatically or even cytolytically on tumor cells (see Evans et al., Immunopharmacology 3, 347-359 (1981)) and c) increase the sensitivity of tumor cells to the organism's cellular immune defense (see Evans et al., Cell. Immunol. S_ 3, 1-15 (1981) and

Ransom et al., Int. J. Cancer 29, 451-458 (1982)).

Furthermore, it is known from the literature that lymphotoxin preparations can lead to regression of tumors in experiments with animals (see Khan et al., Proe. Soc. Exptl. Biol. Med. 169, 291-294 (1982)). Lymphotoxin therefore has of great importance for the prevention and treatment of malignant diseases.

The methods for extracting lymphotoxins that have been made known up to now, in most cases, start from primary cells, e.g. from human peripheral blood or tonsil leukocyte preparations (see Williams et al., J. Immunol. 130, 518-520 (1983)), from mouse spleen cells (see Aksamit et al., Infect. Immun. 3$, 1028-1035 (1982) and Trivers et al., J. Immunol. 117, 130-135 (1976)) or from leukocytes from the abdominal cavity of Syrian hamsters or guinea pigs (see for example Evans et al., Int. J. Cancer 2J, 45-49 (1981)). Here, the cells must first be initiated into the production of lymphotoxins by incubation with high-molecular-weight mitogens, e.g. phytohemagglutinin. Then in the culture fluid from B-lymphocyte cell lines previously only a small amount of lymphotoxin activity could be detected (see Granger et al., J. Immunol. 104, 1476 (1970) and Amino et al., J. Immunol. 113, 1334-1345 (1974)).

Since the previously used methods for the production of lymphotoxins could only be produced in small quantities and only in poor purity, the lymphotoxins produced so far could not be made available either in a form purified to homogeneity or in sufficiently large quantities for extensive investigations of the antineoplastic effects in animal experiments. Furthermore, no biologically active lymphotoxin mRNA could previously be detected either; however, this is a prerequisite for the molecular cloning of the lymphotoxin gene using genetic engineering methods.

The present invention was thus based on the task of producing lymphotoxin and lymphotoxin mRNA in a sufficiently large quantity and purity.

According to the invention, it has now been found that cells of the so-called T-lymphocyte type in monkeys, especially the genus Saguinus,

for example the cell lines L-77/5, A651, A2543, 70N2, 1022 and 1670, preferably the cell lines 1022 and 1670, which upon infection with viruses, preferably with herpes viruses such as herpes virus saimiri or herpes virus ateles, are transformed in vitro or in vivo and made suitable for permanent cultivation in culture, produces significant amounts of lymphotoxin and lymphotoxin mRNA spontaneously, i.e. without further induction, in a suitable culture medium.

Furthermore, it was found that by adding a so-called stimulator to the culture medium, e.g. a tumor promoter known from the literature as a diterpene derivative, preferably of the tiglyan or dafne type, e.g. mezerein or an ester of phorbol, preferably 12-0-tetradecanoylphorbol-13-acetate (see Diamond et al., Advances in Cancer Research Vol. 32, pages 1-74, Academic Press 1980 and Hecker, Carcinogenesis Vol. II, Mechanisms of Tumor Promotion and Cocarcinogenesis, pages 11-48, Raven Press, New

York 1978), at a concentration of 1-1000 ng/ml, the production of lymphotoxin and of lymphotoxin mRNA was further increased.

As culture media, the usual serum-free and serum-containing culture media come into consideration, for example "Eagle's Minimum Essential Medium" or "Roswell Park Memorial Institute Medium 1640" (RPMI1640), to which fetal calf serum and antibiotics can optionally be added, preferably up to 10% serum from calf fetuses, penicillin and streptomycin.

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

In the production of lymphotoxin mRNA, the method according to the invention is preferably carried out in such a way that transformed cells are incubated in a serum-containing culture medium, e.g. e.g. in RPMI 1640 and with 10% fetal calf serum, for 6-48 hours, preferably 12-24 hours, after which the cells are separated and mRNA is isolated in a known manner after destruction of the cells.

In the isolation of the thus formed cell culture product, for the purification of the thus obtained lymphotoxin, chromatography over pore-controlled glass was used for the first time, and the extraction of lymphotoxin mRNA, for example with phenol after destruction of the cell membrane and removal of the cell nucleus , proved particularly suitable.

The further purification of the lymphotoxin takes place, for example, by means of HPLC.

Lymphotoxin with a molecular weight of

60,000 _+ 15% is suitable for preventing and combating tumor cells, e.g. in an aqueous isotonic solution.

The method according to the present invention has the following advantages compared to the state of the art:

1. A homogeneous cell population with unlimited viability is used where propagation can be expanded to any scale, 2. lymphotoxin production in these cultures occurs spontaneously, i.e. no high molecular weight mitogenic substances are added, 3. the production of lymphotoxin can be further increased by addition of low molecular weight stimulants, 4. the production of lymphotoxin can take place in serum-free medium with high yields; you consequently get an excellent starting material for the further protein chemical purification and 5. the RNA isolated from the cells shows immediately detectable lymphotoxin mRNA activity and thereby constitutes an excellent starting material for the further enrichment of this mRNA, which

again constitutes an essential prerequisite for the molecular cloning of mRNA. The cloned monkey lymphotoxin mRNA can again be used as a test compound for the isolation of the homologous human lymphotoxin mRNA or the corresponding gene according to known methods.

The following examples shall explain the present invention in more detail, however without limiting it:

EXAMPLE 1

Production of lymphotoxin using herpesvirus-transformed monkey T-lymphocyte cell lines

The lymphoid cell lines used were incubated in "Roswell

Park Memorial Institute Medium 1640" (RPMI 1640) supplemented with 10% fetal calf serum, penicillin and streptomycin in stationary culture at 37°C under an atmosphere of 95% air and 5% carbon dioxide, and diluted three times weekly with fresh medium .In all experiments, high cell density cultures were diluted 1:2 to 1:3 with fresh medium, on the next day diluted 1:3 once more and occasionally 5 ml was transferred to a culture flask.The culture fluid was recovered by centrifugation after 3 days, frozen and stored at -20°C until testing.

The table below contains the lymphotoxin activities found in three different culture media for each cell line:

Literature references:

A = Fleckenstein et al., Int. J. Cancer 19, 546-554 (1977)

B = Abb et al., Cancer Immunol. Immunother. 9, 219-226 (1980) C = Falk et al., Bacteriol. Pro. 38, 191 (1972)

D = Johnson et al., Proe. Nati. Acad. Pollock. USA 78, 6391-6395

(1981) E = Kaschka-Dierich et al., J. Virol. 44, 295-310 (1982) Review article: Fleckenstein, Biochem. Biophys. Acta 560, 301-342 (1979).

For the production of larger amounts of cells, stirred cultures were used instead of stationary cultures. For that, the cells (for example, the cell line 1670) were cultured, for example, in RPMI 1640 medium with the addition of 10% fetal calf serum in

1- or 2-liter Erlenmeyer flasks on rotary shakers (0.5 1 culture per 1-liter flask; 1 liter culture per 2-liter flask; 40 revolutions per minute; 37°C in normal atmosphere) and diluted in ratio 1:2 to 1:3 three times weekly with fresh culture medium. In this way, the production of culture liquid in the order of 10-100 1 per week is easily possible.

The production of lymphotoxin in 16 70 cells was rather stable over long periods of time: after 9 months in continuous culture, no significant reduction in lymphotoxin activity could be determined in the culture fluids.

Characterization of lymphotoxin:

a) For the biological detection of lymphotoxin activity, the following detection method was used (modified from Trivers et al., J. Immunol. 117, 130-135 and Evans, Cell. Immunol. 63, 1-15 (1981)): Transformed mouse cells of the cell line L929 was 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 with approx. 30,000 cells were pipetted into culture dishes (diameter 16 mm) and incubated for 3-6 hours. Serial dilutions were then carried out on the samples to be tested and incubated for 3 days (all incubations at 37°C).

Finally, the radioactivity in the culture liquid was measured in a liquid scintillation counter.

Fig. 1 shows the detection of lymphotoxin activity in a culture fluid from 1670 cells. Each dilution was tested in parallel in 4 culture dishes. Only the average values were indicated, the standard deviation was 3-4% for the first three dilutions, 6% for the highest dilution. The culture fluid used in this experiment was frozen in more than a hundred ampoules and used as a reference standard in all the lymphotoxin samples; its activity was arbitrarily set at 100 reference units per milliliters (RE/ml).

For comparison, culture fluids from a number of human T-lymphocyte cell lines (MOLT-4, 1301, JURKAT, Karpas-45, CCRF-CEM and CCRF-HSB-2) were examined in the above-mentioned biological lymphotoxin test. In none of the cases could a significant increase in the release of 3H-thymidine be detected compared to untreated controls.

b) The physico-chemical characterization of lymphotoxin activity in culture fluid from cell line 1670 was carried out using

of the following trials:

Stability against heat stress

Stability against acidic pH value

Stability against freezing/thawing

Stability to sodium dodecyl sulfate (SDS)

Stability towards reducing agents (2-mercaptoethanol) Molecular weight determination by high pressure liquid chromatography (HPLC)

The following table shows the result of the stability test:

The activity is very stable against mercaptoethanol, against heating and several periods of freezing/thawing, and is completely destroyed at pH 2 within 24 hours.

The tests regarding heat and pH stability as well as stability against freezing/thawing were carried out with the standard culture fluid from untreated 16 70 cells in the presence of 10% fetal calf serum. The other two experiments were carried out with a culture fluid that was concentrated and enriched over pore-controlled glass (see example 4) from mezerein (20 ng/ml; see example 4) treated 1670 cells (protein content 1-2 mg/ml), which after the incubation with SDS and mercaptoethanol, respectively, were diluted 50 times with complete culture medium (10% calf serum).

The molecular weight of lymphotoxin from 16 70 cells was determined

by high-pressure liquid chromatography (HPLC - see fig. 2).

200 microliters of a pore-controlled glass concentrated culture fluid from untreated 1670 cells was separated by HPLC on a WATERS facility (2 columns 1-125; 20 mM sodium phosphate pH 7, 1 M NaCl, 0.1% "Tween 20" (polyoxy-ethylene-sorbitan-monolaurate, n = 20, molecular weight: approx. 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-inhibitory effect of culture fluids from cell line 1670 was investigated on a number of transformed (tumour-)

cell lines. In these experiments, the tumor cells (grown in "Eagle<1>'s Minimum Essential Medium" with 10% fetal calf serum, penicillin and streptomycin) were placed in culture dishes (50,000 cells per dish with a diameter of 3 cm) and 1 hour later medium or standard was added - the culture fluid from cell line 16 70 (final concentration 16 RE/ml; 3 ml/dish). The cell count per dish was determined after 3 or 4 days of incubation at 37°C (in all experiments, lymphotoxin and medium were added to 2 dishes in parallel). For three cell lines, cell counts were determined daily. The results are reproduced in the following table and in fig. 3:

This shows that culture fluid from 16 70 cells can inhibit the proliferation of tumor cells in different species. This property is characteristic of lymphotoxin (Evans, Cancer Immunol. Immunother.. 12, 181-190 (1983)); it separates this group of proteins in particular from interferons, which can likewise inhibit the growth of tumor cells, but which are usually species-specific, as human interferon in particular is very little or not at all active against mouse cells.

The above-mentioned transformed T-lymphocyte cell lines from humans as well as the (tumor) cell lines are known from the literature.

Cell line 1670 was deposited on 28 March 1984 in accordance with the rules

28 of the European Patent Convention under number 1-294 at "Collection nationale de cultures de microorganismes (C.C.C.M.), Institut Pasteur, Paris".

Example 2

Enhancement of lymphotoxin production in monkey T-lymphocyte cell lines by diterpene compounds

High cell density cultures were diluted 1:2 to 1:3 with fresh medium, diluted 1:3 again the next day and the same stock culture was distributed into three flasks of 5 ml each. The stimulant was added (stock solution 100 micrograms per milliliter in dimethylsulfoxide) t and the cultures were incubated for 3 days. The cells were then removed by centrifugation, the supernatant was frozen and stored at -20°C until the lymphotoxin test.

The following table shows the effect of two selected diterpene tumor promoters, namely 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and mezerein, on lymphotoxin production in selected monkey T-lymphocyte cell lines:

Example 3

Production of lymphotoxin in serum-free medium

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

The table below shows a comparison between lymphotoxin production in 16 70 cells analogous to example 1 in serum-containing and serum-free medium in the presence of 20 ng mezerin/ml:

The protein determination was carried out according to the method of Bradford (see Anal. Biochem. 72, 298 (1976)).

Example 4

Concentration and purification of lymphotoxin from 16 70 cells by chromatography over pore-controlled glass columns 16 70 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 speed 40 revolutions per min.). Cells from well-growing cultures were recovered by centrifugation, washed and resuspended in serum-free RPMI-1640 medium supplemented with 20 ng/ml mezerein. The suspension was stored for 3 days in 150 cm 2 plastic culture bottles (200 ml per bottle), then the culture liquid was recovered by centrifugation and filtration.

The culture liquid thus prepared was chromatographed over 10 ml of pore-controlled glass (pore diameter: 350 Angstroms, particle size: 120-200 mesh; column diameter: 9 mm, flow rate: 140 ml/hour). The column was then washed with 10 mM sodium phosphate buffer pH 7.2/1.5 M NaCl and finally eluted with the same buffer in 50% ethylene glycol (vol/vol).

The following table reproduces the results of two experiments carried out in this way:

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 with

10% fetal calf serum at a cell density of approx. 5 x 10 5 cells/ml and added 20 ng mezerin/ml. 20 hours later, the cells were harvested by centrifugation, washed in an appropriate buffer (e.g. 10 mM Tris.HCl pH 7.4, 140 mM NaCl, 1.5 mM MgCl2) and suspended in 10 ml of lysis buffer (e.g. 10 mM Tris.HCl pH 8.4, 140 mM NaCl, 1.5 mM MgCl-, 0.5% "Nonidet-P 40" [Nonionic Detergent ]). After 10 minutes of incubation in an ice bath, the suspension was shaken for a short time and the cell nuclei removed by centrifugation. From the supernatant from the centrifugation (= cytoplasm), RNA was isolated by a method known per se, e.g. by extraction with phenol (see Aviv et al., Proe. Nati. Acad. Sei. USA 69, 1408-1412 (1977)). RNA became

finally dissolved in water (concentration approx. 3-5 mg/ml) and

translated in oocytes Xenopus laevis by a method already known per se (see for example Colman et al., Cell 17, 517-526 (1979), Hitchcock et al., Anal. Biochem. 109, 338-344

(1980) and Contreras et al., Anal. Biochem. 113, 185-187 (1981)). The translation products were subsequently checked in the biological test for their lymphotoxin activity.

The following table shows the cytotoxic activity found in oocyte culture fluid compared to RNA preparations from M0LT-4 cells, a human T-lymphocyte cell line whose culture fluids show no detectable lymphotoxin activity, and culture fluids from untreated oocytes:

The table shows that all three RNA preparations from 16 70 cells as well as an RNA preparation from 1022 cells in up to 14 7-fold dilution cause a significant increase in released radioactivity, while RNA from M0LT-4 cells does not show any such activity . These experiments show that biologically active lymphotoxin mRNA is found in RNA preparations from 1670 and 1022 cells.

Claims (13)

1. Method for the production of lymphotoxin or lymphotoxin mRNA, characterized in that monkey T-lymphocytes that have been transformed with a virus are grown in a suitable medium in continuous culture and that the lymphotoxin or lymphotoxin mRNA is isolated according to methods known per se. 2. Method according to claim 1, characterized in that herpes virus is used as virus, preferably herpes virus saimiri or herpes virus ateles. 3. Method according to claim 1, characterized in that cells from the genus Saguinus are used as monkey T-lymphocytes. 4. Method according to claim 3, characterized in that the cell line 1022, 1670, 70N2, L77/5, A651 or A 2543 is used as the T-lymphocyte cell line. 5. Method according to claim 3. characterized in that the cell line 1022 or 1670 is used as the T-lymphocyte cell line. 6. Method according to claims 1 to 5, characterized in that up to 10% calf tissue cheese serum is added to the culture medium. 7. Method according to claims 1 to 6, characterized in that low molecular weight tumor promoters are added to the culture medium for production stimulation, preferably diterpenes, particularly of the daphne, ingelan and tiglian type, or indole alkaloids. 8. Method according to claims 1 to 7, characterized in that an ester of phorbol or mezerein is added as a tumor promoter in a concentration of 1-1000 ng/ml, preferably 10-100 ng/ml, to stimulate lymphotoxin production during the incubation. 9. Method according to claims 1 to 7, characterized in that an ester of phorbol or mezerein is added as a tumor promoter in a concentration of 1-1000 ng/ml, preferably 10-100 ng/ml, to stimulate lymphotoxin production during the incubation. 10. Method according to claims 1 to 8, characterized in that the cells are transferred to cell-free medium after propagation of the cells in serum-containing medium. 11. Method according to claims 1 to 8 and 10. characterized in that the lymphotoxin is separated from the culture fluid after 1 to 7 days, preferably after 2 to 4 days. 12. Method according to claims 1 to 8, 10 and 11, characterized in that the lymphotoxin is enriched over a column with pore-controlled glass. 13. Method according to claims 1 to 7 and 9, characterized in that the cells are incubated in a culture medium containing fetal calf serum, and that lymphotoxin mRNA is isolated from the cells after 3 to 48 hours, preferably after 6 to 24 hours.