DE3417277A1 - Process for the preparation of lymphotoxin and of lymphotoxin mRNA - Google Patents

Abstract

The present invention relates to a process for the preparation of lymphotoxin and lymphotoxin mRNA by incubation of transformed T lymphocytes in permanent culture and subsequent removal of the cell products formed in this way.

Description

Process for the production of lymphotoxin and of

Lymphotoxin mRNA From the literature it is known that certain cells of mammals, after suitable stimulation in vitro, are capable of so-called Produce lymphotoxins (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). Here, lymphotoxin represents a given Species are not a uniform protein in vitro; rather, they are a series of Described subtypes that differ in molecular weight, charge and stability (see, for example, Granger et al. in Biology of the Lymphokines pp. 141-180, Academic Press 1979). For example, there are four classes of lymphotoxins in the human system distinguished, their molecular weights in the ranges from 10,000 to 20,000 for yLT, 35,000 to 50,000 for ßLT, 70,000 to 90,000 for aLT and 200,000 to 600,000 for LT complex lie.

Lymphotoxins are capable of a) malignant transformation in vitro of cells (see Evans et al. in Int. J. Cancer 27, 45-49 (1981) and Ransom et al. in J. Natl. Cancer Inst. 69, 741-744 (1982)), b) to act cytostatically or even cytolytically on tumor cells (see Evans et al. in Immunopharmacology 3, 347-359 (1981)) and c) the sensitivity of tumor cells to increase the cellular immune defense of the organism (see Evans et al. in Cell. Immunol. 63, 1-15 (1981) and Ransom et al. in Int. J. Cancer 29,451-458 (1982)).

It is also known from the literature that lymphotoxin preparations can also induce tumor regression in animal models (see Khan et al. in Proc. Soc. Exptl.

Biol. Med. 169: 291-294 (1982)). Lymphotoxins are therefore of great importance for the prophylaxis and therapy of malignant diseases.

The previously known method for the production of lymphotoxins in most cases start from primary cells, e.g. from leukocyte preparations from human peripheral blood or tonsils (see Williams et al. in J. Immunol. 130, 518-520 (1983)), from spleen cells of mice (see Aksamit et al. In Infect. Immune. 36, 1028-1035 (1982) and Trivers et al. in 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. in Int. J. Cancer 27: 45-49 (1981)). Here these cells must first be incubated with high-molecular mitogens, e.g. phytohemagglutinin, are stimulated to produce lymphotoxins. So far in the B-lymphocyte cell lines survived only low levels of lymphotoxin activities in culture can be determined (see Granger et al.

in J. Immunol. 104, 1476 (1970) and Amino et al. in J.

Immunol. 113: 1334-1345 (1974)).

Because according to the methods used so far for the production of lymphotoxins could only be produced in small quantities and in only low purity the ones produced so far Lymphotoxins neither up to homogeneity cleaned in sufficient quantities for more extensive antineoplastic examinations Effect on the animal model are made available. In addition, so far also no biologically active lymphotoxin mRNA can be detected; This is however the prerequisite for the molecular cloning of the lymphotoxin genes means of the methods of genetic engineering.

The present invention was therefore based on the object of lymphotoxin and to produce lymphotoxin mRNA in sufficient quantity and purity.

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, 70N2, 1022 and 1670, but preferably cell lines 1022 and 1670, caused by infection with viruses, preferably with herpes viruses such as Herpes virus saimiri or Herpes virus ateles, transformed in vitro or in vivo and to permanent growth in culture were enabled spontaneously in a suitable culture medium, that is to say without further induction, produce significant amounts of lymphotoxin and lymphotoxin mRNA.

It was also found that by adding a so-called Stimulator for Culture medium, e.g. a tumor promoter known from the literature such as a diterpene derivative, preferably of the Tiglian or Daphnan type, e.g. Mezerein or an ester of Phorbols, preferably 12-0-tetradecanoylphorbol-13-acetate (see Diamond et al. in Advances in Cancer Research Vol. 32, pages 1-74, Academic Press 1980 and Hecker in Carcinogenesis Vol. II, Mechanisms of Tumor Promotion and Cocarcinogenesis, pp 11-48, Raven Press, New York 1978), at a concentration of 1-1000 ng / ml die Production of lymphotoxin and of lymphotoxin mRNA is further increased.

The usual serum-free and serum-containing media are used as culture media Culture media into consideration, for example Eagle's Minimum Essential Medium or Roswell Park Memorial Institute Medium 1640 (RPMI1640) each of which was fetal calf serum and antibiotics can be added, preferably up to 10% fetal calf serum, Penicillin and streptomycin.

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

During the production of the lymphotoxin mRNA, the The method is preferably carried out in such a way that transformed cells after addition from 10-100 ng / ml 12-0-tetra-decanoylphorbol-13-acetate or mezerein as a stimulator in a culture medium containing serum, e.g. in RPMI1640 and 10% fetal calf serum 6-48 hours, preferably 12 to 24 hours, then the Cells are separated and after destruction of this the mRNA according to known Procedure is isolated.

During the isolation of the cell culture products formed in this way, the purification of the lymphotoxin obtained in this way, the chromatography via pore-controlled Glass, which first was applied, and, after destruction of the Cell membranes and removal of the cell nuclei, extraction of the lymphotoxin mRNA for example with phenol proved to be particularly suitable.

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

The lymphotoxin thus obtained, having a molecular weight of 60,000 + 15% is, e.g. in an aqueous isotonic solution, for prophylaxis and control suitable for tumor cells.

Compared to the prior art, the method according to the invention the following advantages: 1. It becomes a homogeneous cell population with unlimited Viability used, the reproduction of which can be expanded on any scale 2. the lymphotoxin production in these cultures occurs spontaneously, that is no high molecular weight mitogens have to be added, 3. the production of Lymphotoxin can be further increased by low molecular weight stimulators, 4. the production of lymphotoxin can take place in serum-free medium with high yields; one thus obtains an excellent starting material for the further protein chemical Purification and 5. the RNA isolated from the cells shows immediately detectable Lymphotoxin mRNA activity, making it an excellent starting material for the further enrichment of this mRNA, which in turn is an essential prerequisite for represents the molecular cloning of the mRNA. The cloned monkey lymphotoxin mRNA can in turn by known methods as a sample for the isolation of the homologous human lymphotoxin mRNA or the corresponding genes can be used.

The following examples are intended to explain the present invention in more detail. but without limiting it: Example 1 Production of lymphotoxin by herpesvirus-transformed Af fen T lymphocyte cell in ien The lymphoid cell lines used were in Roswell Park Memorial Institute Medium 1640 (RPMI 1640) with added 10% fetal Calf serum, penicillin and streptomycin in stationary culture at 370C in one atmosphere Made of 95% air and 5% carbon dioxide and kept fresh three times a week Medium diluted. In all experiments, dense cultures were made with fresh medium Diluted 1: 2 to 1: 3, the next day again diluted 1: 3 and each 5 ml in attached to a culture flask. The culture supernatants were through after three days Obtained by centrifugation, frozen and stored at -200C until the test.

The following table contains the lymphotoxin activities determined in three different culture supernatants of each cell line: Cell line origin reference lymphotoxin activity (transforming RE / ml Herpes virus = HV) Expt. 1 expt. 2 expt. 3 L-77/5 Saguinus oedipus - lymph nodes A 1 3 1 of a tumor-bearing animal (HV saimiri) A 651 Saguinus oedipus - in vitro Trans- B 15 30 25 formation of lymphocytes (HV ateles) A 2543 Saguinus oedipus - in vitro trans- B 90 56 48 formation of lymphocytes (HV ateles) 70N2 Saguinus oedipus - thymus A, C, D, E 56 47 50 of a tumor-bearing animal (HV saimiri) 1022 Saguinus oedipus - tumor D 580 480 630 (HV ateles) 1670 Saguinus nigricollis - spleen A, C, E 88 83 77 of 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 = Falk et al., Bacteriol. Proc. 38: 191 (1972) D = Johnson et al., Proc. Natl. Acad. Sci. 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 quantities of cells, moving cultures were used instead of stationary cultures. For this purpose, the cells (for example from line 1670) were grown, 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 l flask; 1 1 Culture per 2 l flask; 40 revolutions per minute; 370C 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 magnitude of 10-100 liters per week is easily possible.

The production of lymphotoxin in 1670 cells was over long periods of time stable over time: after 9 months in continuous culture was none significant Decrease in lymphotoxin activity in the culture supernatants detectable.

Characterization of the lymphotoxin: a) For the biological detection of The following detection method was used for lymphotoxin activity (modified from Trivers et al. in J. Immunol. 117, 130-135 and Evans in Cell.

Immunol. 63, 1-15 (1981)): Transformed mouse cells of the cell line L929 were incubated overnight with 1 microCurie 3H-thymidine / ml culture medium (preferably Eagle's Minimum Essential Medium with 10% fetal calf serum) cultured, then trypsinized and suspended in fresh culture medium containing 5% fetal calf serum. 0.5 ml each of this Suspensions with about 30,000 cells were pipetted into culture dishes (diameter 16 mm) and incubated for 3-6 hours. This was followed by serial dilutions of the to be tested Sample applied and incubated for three days (all incubations at 370C). The radioactivity the culture supernatants were finally measured in a liquid scintillation counter.

The following figure shows the evidence of lymphotoxin activity of a culture supernatant from 1670 cells.

Each dilution was tested in parallel in 4 culture dishes.

Only the mean values were given; 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 ampoules and used as a reference standard in all lymphotoxin tests; its activity was arbitrarily assumed to be 100 reference units per milliliter (RU / ml): Culture supernatant from 1670 cells controls (medium) For comparison, culture supernatants from a number of human T lymphocyte cell lines (MOLT-4, 1301, JURKAT, Karpas-45, CCRF-CEM and CCRF-HSB-2) were used in the biological Lymphotoxin test studied. In no case was there a significant increase in 3H-thymidine release compared to untreated controls.

b) The physicochemical characterization of the lymphotoxin activity in culture supernatants of the cell line 1670 was carried out by the following experiments: Stability against thermal stress Stability against acidic pH value Stability against freezing / thawing Stability against sodium dodecyl sulfate (SDS) Stability against reducing agents (2- Mercaptoethanol) Molecular weight determination by high pressure liquid chromatography (HPLC) The following table shows the results of the stability tests: Treatment of lymphotoxin activity in Percent of control worth 560C - 5 hours 68% 5 freeze / thaw cycles 108% pH 2.0 - 24 hours at 40C 1% 0.1% SDS - 1 hour at 370C 15% 0.1 M 2-mercaptoethanol - - 1 hour at 370C 95% The activity is very stable against mercaptoethanol, against heating and several cycles of freezing / thawing, and is completely destroyed within 24 hours at pH 2.

The experiments on thermal and pH stability as well as opposite Freeze / thaw were performed with the standard culture supernatant from untreated 1670 cells in the presence of 10% fetal calf serum performed. The others Both experiments were carried out with a pore-controlled glass (see example 4) concentrated and enriched culture supernatant from mezerein (20 ng / ml; see example 4) treated 1670 cells carried out (protein content 1-2 mg / ml), that after incubation with SDS or mercaptoethanol with complete culture medium (10% calf serum) has been diluted 50 times.

The molecular weight of the lymphotoxin from 1670 cells was determined by high pressure liquid chromatography (HPLC - see figure below): 200 microliters of a culture supernatant from untreated 1670 cells concentrated through pore-controlled glass were separated by means of HPLC on a system from WATERS (2 columns I-125; 20 mM sodium phosphate pH 7, 1 M 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 to determine the molecular weight, serum albumin, ovalbumin, trypsinogen and lysozyme separated in the same system. The analysis showed a single peak of activity at a molecular weight of 60,000 + 15%.

c) The growth-inhibiting effect of culture supernatants of the cell line 1670 was investigated on a number of transformed (tumor) cell lines. In these experiments, the tumor cells (cultivated in Eagle'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 a few hours later with medium or the standard culture supernatant of the Cell line 1670 added (final concentration 16 RU / ml; 3 ml / dish). After 3 or 4 days of incubation at 37 ° C., the number of cells per dish was determined (in all experiments two dishes were prepared in parallel with lymphotoxin or medium). The number of cells was determined daily in three cell lines. The results are shown in the following table or figure: Species Cell line Incubation time Cell count / 3 cm culture dish (Days) (10E-5 x cells / dish) Control lymphotoxin Human Hela 4 8.1 0.6 Hep-2 3 9.3 2.1 A-549 4 10 3.4 Monkey GL-V3 3 5.6 4.5 Vero 3 6.3 5.3 Mouse L929 4 8.5 3.1 IT22 3 13 4.0 B16 4 19 4.9 Rabbit RK13 3 4.6 3.5 Controls Lymphotoxin These show that culture supernatants from 1670 cells can inhibit the multiplication of tumor cells of various species.

This property is characteristic of lymphotoxins (Evans in Cancer Immunol. Immunother. 12: 181-190 (1983)); it distinguishes this group of proteins in particular of the interferons, which are also the growth of Can inhibit tumor cells, but are generally species-specific, wherein especially human interferon very little or not at all active on mouse cells are.

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

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

Example 2 Increase in Lymphotoxin Production in Monkey T Lymphocyte Cell Lines by diterpene compounds Dense cultures were replenished with fresh medium 1: 2 to 1: 3 diluted, diluted again 1: 3 the next day and each time 5 ml of the same stock culture divided into three vials. The stimulators were added (stock solutions 100 micrograms / ml in dimethyl sulfoxide) and the cultures were incubated for three days. The cells were then removed by centrifugation and the supernatants frozen and stored at -200C 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: Cell line experiment concentration lymphotoxin activity in the supernatant (RE / ml) (ng / ml) Control TPA 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 1 100 480 760 930 2 100 630 700 610 Example 3 Production of Lymphotoxin in Serum-Free Medium After the cells had proliferation in serum-containing medium (analogous to Example 2), the cells were transferred to serum-free medium.

The following table shows a comparison of the lymphotoxin production in 1670 cells analogous to Example 1 in serum-containing and serum-free medium in the presence of 20 ng mezerein / ml: Experi- serum content lymphotoxin protein spec. ment of the medium activity content activity I%) (RE / ml) (mg / ml) vity (RE / mg) 1 10 370 5 74 2 10 180 5 36 3 10 270 5 54 4 0 140 0.050 2.800 5 0 72 0.053 1.400 6 0 160 0.044 3.700 The protein determination was carried out by the Bradford method (see Anal. Biochem. 72, 298 (1976)).

Example 4 Concentration and Purification of Lymphotoxin from 1670 cells 1670 cells were obtained by chromatography on columns of pore-controlled glass grown in RPMI-1640 medium with 10% fetal calf serum in rotating Erlenmeyer flasks (0.5 l culture in a 1 l flask, rotation speed 40 revolutions per minute). Cells from well growing cultures were collected by centrifugation, washed and resuspended in serum-free RPMI-1640 medium with the addition of 20 ng / ml mezerein. The suspension was made into three 150 cm2 plastic culture bottles (200 ml per bottle) Held stationary for days, then the culture supernatant was centrifuged and filtration recovered.

Culture supernatants produced in this way were pore-controlled over 10 ml Chromatographed glass (pore diameter: 350 Angstroem, grain size: 120-200 mesh; Column diameter: 9 mm, flow rate: 140 ml / hour). Subsequently was the column was 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 (v / v).

The following table gives the results of two such experiments: Volume of lymphotoxin protein specific activity (ml) (Re / ml)% (mg / ml) RE / mg Experiment 1 Sample 2,800 162 100 0.044 3,700 Throughput 2,800 13 8 - - Washing fraction 10 96 1 - - Eluate 20 17,300 76 2.0 8,650 Experiment 2 Sample 2,500 141 100 0.050 2,800 Eluate 17 8,600 41 1,6 5,400 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 about 5 × 10 5 cells / ml and treated with 20 ng of mezerein / ml offset. 20 hours later, the cells were harvested by centrifugation, washed in a suitable buffer (for example 10 mM Tris.HCl pH 7.4, 140 mM NaCl, 1.5 mM MgCl2) and in 10 ml of lysis buffer (for example 10 mM Tris. HCl pH 8.4, 140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet-P 40 [nonionic detergent, trademark of SHELL] After ten minutes of incubation in an ice bath, the suspension was shaken briefly and the cell nuclei were removed by centrifugation.

The centrifugation supernatant (= cytoplasm) became through an known processes, for example by extraction with phenol (see Aviv et al. in Proc. Natl. Acad. Sci.

USA 69, 1408-1412 (1977)) isolated the RNA. She eventually became dissolved in water (concentration about 3-5 mg / ml) and by means of already known ones Method translated into Xenopus laevis - oocytes (see for example Colman et al. in Cell 17: 517-526 (1979), Hitchcock et al. in Anal.

Biochem. 109: 338-344 (1980) and Contreras et al. in Anal.

Biochem. 113: 185-187 (1981)). The translation products were in biological test checked for their lymphotoxin activity.

The following table shows the cytotoxic activity found in oocyte supernatants compared to RNA preparations from MOLT-4 cells, one human T lymphocyte cell line whose culture supernatants have no detectable lymphotoxin activity as well as supernatants from untreated oocytes: preparation Radioactivity in the supernatant (cpm / 100 microliter) Dilution of the oocyte supernatants 1: 12 1: 42 1: 147 1: 514 controls (uninjected oocytes) 2,200 2,300 2,100 2,500 2,400 2,200 1,800 3,000 MOLT-4 RNA Expt. 1 1,600 2,400 1,700 2,200 Expt. 2 1,700 2,300 1,600 1,800 1670-RNA Expt. 1 4,700 4,700 3,600 2,800 Exp. 2 4,300 3,800 3,300 2,600 Expt. 3 3,200 2,800 2,800 controls 4,400 3,300 - -1670-RNA Expt. 1 6,100 5,000 - -1670 RNA Expt. 2 6,600 5,100 - -1022-RNA Expt. 1 7,000 5,000 - - The table shows that all three RNA preparations from 1670 cells as well as an RNA preparation from 1022 cells in up to 147-fold dilution one caused a significant increase in released radioactivity while RNA from MOLT-4 cells does not have such activity. These experiments show that in RNA preparations from 1670 and 1022 cells biologically active lymphotoxin mRNA is included. - L e r s e i t e -

Claims (18)

PATENT CLAIMS r9 process for the production of lymphotoxin or Lymphotoxin mRNA, characterized in that monkey T lymphocytes transformed with viruses be grown in a suitable medium in permanent culture and the lymphotoxin or the lymphotoxin mRNA is isolated by methods known per se. 2. The method according to claim 1, characterized in that as viruses Herpesviruses, but preferably Herpesvirus saimiri or Herpesvirus ateles, used will. 3. The method according to claim 1, characterized in that the monkey T lymphocytes Cells from the genus Saguinus can be used. 4. The method according to claim 3, characterized in that the T-lymphocyte cell lines lines 1022, 1670, 70N2, L77 / 5, A651 or A 2543 can be used. 5. The method according to claim 3, characterized in that the T-lymphocyte cell lines lines 1022 or 1670 can be used. 6. Process according to claims 1-5, characterized in that up to 10% fetal calf serum is added to the culture medium. 7. Process according to claims 1-6, characterized in that the culture medium for stimulating production of low molecular weight tumor promoters, preferably Diterpenes, in particular of the Daphnan, Ingelan and Tiglian types, or indole alkaloids can be added. 8. The method according to claims 1 to 7, characterized in that that to stimulate the production of lymphotoxin during incubation as a tumor promoter an ester of phorbol or mezerein, at a concentration of 1-1000 ng / ml, preferably but from 10-100 ng / ml, is added. 9. The method according to claims 1 to 7, characterized in that that to stimulate the production of lymphotoxin mRNA during incubation as Tumor promoter an ester of phorbol or mezerein, in a concentration of 1-1000 ng / ml, but preferably from 10-100 ng / ml, is added. 10. The method according to claims 1 to 8, characterized in that that after proliferation of the cells in serum-containing medium, the cells in serum-free Medium to be transferred. 11. The method according to claims 1 to 8 and 10, characterized in that that the lymphotoxin from the culture supernatants after 1 to 7 days, but preferably after 2 to 4 days, is separated. 12. The method according to claims 1 to 8, 10 and 11, characterized in that that the lymphotoxin is enriched over a column of pore-controlled glass. 13. The method according to claims 1 to 7 and 9, characterized in that that the cells are incubated in a culture medium containing fetal calf serum and the lymphotoxin mRNA from the cells after 3 to 48 hours are preferred but after 6 to 24 hours, is isolated. 14. Lymphotoxin, producible according to claims 1 to 8 and 10 to 12th 15. Lymphotoxin according to claim 14, characterized in that this has a molecular weight of 60,000 + 15%. 16. Lymphotoxin mRNA, producible according to claims 1 to 7, 9 and 13th 17. Medicaments containing lymphotoxin according to claims 14 or 15th 18. Medicament according to claim 17 for combating tumor cells.