JPH0811759B2 - New lymphokine Ⅱ and its manufacturing method and applications - Google Patents

Description

The present invention relates to a novel lymphokine II having cytotoxic activity against tumor cells, a process for producing the same, and a use thereof.

As lymphokines having cytotoxic activity against tumor cells, lymphotoxins derived from animals including humans, and Tumor necrosis factor derived from animals other than humans (hereinafter referred to as “TNF”) are known.

Lymphotoxin is co-authored by Ryuichi Aoki et al., “Lymphokine”, New Immunology Series 6, pages 87-105 (1979), Medical Institute, Bloom BR and Glade PR, “In Vitro Methods”. In Vitro Methods in Cell-Mediated Immunit
y) ”, Academic Press (197
1 year) and “Cellular Immu
nology) ", Vol. 38, pp. 388-402 (1978), and TNF is described in Carswell E.A.
EA) etc., “Proceedings of the National Academy of Science of the You”
SAS (Proceedings of the National Academy of
Sience of the USA) ", Vol. 72, No. 9, 3666
~ 3670 (1975) and edited by E. Pick, "Tumor Necrosis Factor in Lymphokines", Part 1
Volume II, pp. 235-272, Academic Press (Academic
Press) (1981).

In addition, recently, Haruo Onishi et al. Have disclosed an antitumor glycoprotein, which is a kind of lymphokine, in JP-A-58-146293.

The inventors have been studying lymphokines for many years. As a result, the existence of a new lymphokine II having physicochemical properties not described in the literature, which is completely different from those of the conventionally known lymphokines, was confirmed, a manufacturing method thereof was established, and cytotoxic activity against various malignant tumor cells was recognized, The application was established and the present invention was completed.

That is, the present invention, the physicochemical properties, molecular weight 20,000 ± 2,000 isoelectric point pI = 6.2 ± 0.3 mobility Disc-PAGE, Rf = 0.29 ± 0.02 UV absorption spectrum 280nm maximum absorption solubility in the solvent water, Soluble in physiological saline or phosphate buffer Slightly insoluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Shows a protein positive reaction by the Lowry method or micro burette method, and sugar by the phenol sulfate method or anthrone sulfate method. A cytoplasmic positive action L ₉₂₉ shows cytotoxic activity against ₉₂₉ cells, and virtually no cytostatic or interferon activity against KB cells.Activity stability in aqueous solution Stable up to 60 ℃, 4
Stable in the range of pH 4.0 to 11.0 under the condition of holding at 16 ° C for 16 hours. New lymphokine II that is stable for more than 1 month when stored frozen at -10 ° C (throughout this specification, this substance is referred to as new lymphokine II). And its manufacturing method and uses.

The production method of new lymphokine II is, for example, new lymphokine
A human-derived cell having II-producing ability, for example, leukocytes, lymphocytes, cultured cells, and the like may be produced by inducing an inducer.

Human-derived white blood cells and lymphocytes may be prepared by separating blood collected from human.

As the cells of human origin that have been cultured into culture cells, cells grown in vitro can be used according to a conventional method.

However, in the case of the present invention, during the growth of the cultured cell line, it is directly transplanted into a warm-blooded animal other than human or inoculated into a diffusion chamber to supply the body fluid of the warm-blooded animal. It is desirable to grow while receiving it.

That is, unlike the case of growing in vitro, a nutrient medium containing expensive serum and the like is not necessary or can be greatly saved, and maintenance during cell growth is extremely easy. In addition, the novel lymphokine II activity that is induced and produced from the obtained cells is characteristic.

The method of using warm-blooded animals other than humans is to transplant human-derived cells in culture into warm-blooded animals other than humans, or to use a diffusion chamber that can receive the supply of body fluids from the animals. If the animal is buried in an animal body and then reared normally, the cells thereof can easily grow using the body fluid containing the nutrients supplied from the warm-blooded animal body.

Furthermore, compared with the case of growing in vitro, the growth of these cells is stable, the growth rate is high, the amount of cells obtained is large, and further, the new lymphokine per cell is A significant feature is that the yield of II is significantly increased.

Cultured human-derived cells used in the present invention are
It can be transplanted into warm-blooded animals other than humans and easily proliferate,
Moreover, any cell having the ability to produce new lymphokine II may be used, and for example, various cell lines derived from humans described in “Protein Nucleic Acid Enzyme Vol. 20, No. 6” pages 616 to 643 (1975) should be used. You can Journal of Clinical
Microbiology (Journal of Clinical Microbiolo
gy), Vol. 1, 116-117 (1975), Namalwa cells, I. Miyoshi, Nature, 267, 843-844. (1977
, BALL-1 cells, TALL-1 cells, NA
LL-1 cells, "The Journal of Immunology", Vol. 113, No. 1334-
M-7002 cells, B-7101 cells, "Tissue culture", Vol. 6, No. 13, 527-546 (1980) described on page 1345 (1974).
, JBL cells, EBV-Sa cells, EBV-wa
Cells, EBV-HO cells, MOLT-3 cells, and other BALM-2
Cells, CCRF-SB cells, (ATCC CCL120) CCRF-CEM cells,
Preferred are established lymphoblastoid cells such as DND-41 cells, and cells obtained by treating normal mononuclear cells, granular leukocyte cells, etc. with various viruses, drugs, radiation and the like to establish a culture cell line. Is.

In addition, a gene having the ability to produce new lymphokine II in these human-derived cells, for example, means of cell fusion using polyethylene glycol or Sendai virus,
It can be treated by gene recombination using enzymes such as DNA ligase, restriction enzymes (nucleases), and DNA polymerase to increase its growth rate or enhance the ability to produce new lymphokine II per cell. Often,
It is not limited to only the cell lines described herein. These cells can be freely used alone or in the form of a mixture of two or more kinds in the process of inducing and producing new lymphokine II described later. If necessary, leukocytes, lymphocytes, etc. collected from humans and prepared can be used in combination therewith.

The warm-blooded animals used in the present invention may be those capable of proliferating human-derived cells, for example, birds such as chicken and pigeons, dogs, cats, monkeys, rabbits, goats, pigs, horses,
Mammals such as cows, guinea pigs, rats, nude rats, hamsters, normal mice and nude mice can be used.

Since transplanting human-derived cells into these animals may cause an undesired immune reaction, in order to suppress the reaction as much as possible, the animals used should be in a juvenile state, that is, egg, embryo, fetus, or neonatal period, Those in childhood are preferred.

Further, these animals may be subjected to pretreatment such as irradiation with X-rays or gamma rays of about 200 to 600 rem or injection of antiserum or immunosuppressive agent to weaken the immune reaction and transplant.

If the animal used is a nude mouse or nude rat, the immune reaction is weak even if it is grown, so there is no need for pretreatment of these, and cells of human origin that have been cultured and cultured are transplanted. It is particularly advantageous as it can and can grow rapidly.

In addition, for example, after transplanting cells of human origin in culture into a hamster to proliferate them, and then transplanting the cells into a nude mouse, the cells are transplanted between warm-blooded animals other than humans and humans. More stable growth of the cells of origin,
It is also free to increase the amount of new lymphokine II derived from them.

In this case, not only the same species and the same genus but also the same rope and the same gate may be transplanted. The site in the animal body into which the human-derived cells are transplanted may be any site where the transplanted cells can proliferate, and is freely selected, for example, urinary cavity, vein, abdominal cavity, or subcutaneous site.

Further, without transplanting human-derived cells into the animal body, a porous filtration membrane capable of blocking passage of animal cells, for example, a membrane filter having a pore size of about 10 ^-7 to 10 ^-5 m,
A known diffusion chamber of various shapes and sizes provided with an ultrafiltration membrane or a follow fiber is embedded in the animal body, for example, the abdominal cavity, and the body fluid containing nutrients is supplied from the animal body while the chamber is being supplied. Any of the above-mentioned cultured cells derived from humans can be grown therein.

If necessary, the solution containing the nutrients in the chamber is connected to the body fluid in the animal body, and a chamber for perfusion is attached to, for example, the surface of the animal body to check the growth state of human-derived cells in the chamber. It is also possible to see through a window provided on the surface of the animal, and to make it possible to detach and replace only this chamber part to grow cells with a long life span without killing the animal, thereby increasing the cell production per animal. It can be further increased.

In the method using these diffusion chambers, human-derived cells do not come into direct contact with animal cells, so that not only human-derived cells can be easily collected, but also there is little concern that an undesired immune reaction will occur. It has the feature that various warm-blooded animals can be used freely without the need for pretreatment for suppression.

Maintenance of the transplanted animal is convenient because normal breeding management of the animal may be continued, and no special handling is required even after transplantation.

The period for growing human-derived cells is usually 1 to 10
You can achieve your goals within a week's time. The number of human-derived cells thus obtained is about 10 ⁷ to 10 10 per animal.
We also found that it could reach ¹² or more.

In other words, the number of human-derived cells grown in an animal body reached about 10 ² to 10 ⁷ times the number of cells transplanted per animal individual, or even more, and the number of cells inoculated into an in vitro nutrient medium was increased. Approximately 10 ¹ to 10 ⁶ times, or even more, when grown, is extremely advantageous for the production of new lymphokine II.

Any method can be used to induce and generate new lymphokine II from human-derived cells grown in this manner. It is also possible for the new lymphokine II inducer to act on the grown animal body. For example, human-derived cells that have been grown in suspension in ascites in the abdominal cavity, and tumor cells that have been subcutaneously produced by inducing a new lymphokine II inducer by directly acting, and then the serum, The new lymphokine II may be purified and collected from ascites or tumor.

Alternatively, human-derived proliferating cells can be taken out of an animal body other than human and a new lymphokine II inducer can be caused to act in vitro to induce and produce new lymphokine II. For example, human-derived cells grown in ascites are collected, or tumors containing human-derived cells produced subcutaneously are excised and dispersed, and the obtained cells are concentrated in a nutrient medium kept at about 20 to 40 ° C. To about 10 ⁵ to 10 ⁸ / ml, and a new lymphokine II inducer is allowed to act on this to induce and produce new lymphokine II, which can be purified and collected.

Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells are left in the chamber or removed from the chamber, and a new lymphokine II inducer is acted on to induce and produce new lymphokine II. You can also

In addition, as described above, human-derived cells obtained by using warm-blooded animals other than humans are further cultured in vitro for about 1 to 4 days, if necessary, after synchronizing the growth generation of the cells, and the like. The lymphokine II inducer can be acted on to induce and produce new lymphokine II.

In addition, for example, after inducing new lymphokine II to be produced in the expanded human-derived cells as they are in the animal body, and then ex vivo from the human-derived cells collected from a specific site or whole of the same animal individual. Method for inducing and producing new lymphokine II, or a method in which cells once used for inducing and producing new lymphokine II are used twice or more times for inducing and producing new lymphokine II, or a chamber for embedding or connecting to an animal body It is also free to further increase the amount of new lymphokine II produced per animal individual used by, for example, a method of increasing the number of cells obtained by exchanging.

Examples of the new lymphokine II inducer of the present invention include viruses known as α-interferon inducers, nucleic acids, nucleotides, and phytohemagglutinin known as γ-interferon inducers, concanavalin A, porkweed mitogen, lipoprotein. Polysaccharides, endotoxins, polysaccharides, bacteria and the like are used as appropriate. In addition, for sensitized cells, the antigen is also an inducer of new lymphokine II.

Furthermore, when inducing new lymphokine II from cells of human origin, a new lymphokine II inducer, α-
It is also free to increase the production amount of new lymphokine II by using an interferon inducer and a γ-interferon inducer in combination.

Moreover, it was found that not only the new lymphokine II is produced by these induced productions, but also human interferon with high species specificity is produced at the same time.

This enables simultaneous production of two or more valuable human physiologically active substances, further enables high-level utilization of human-derived cells, and supplies a large amount of new lymphokine II and human interferon at low cost. It is convenient.

The new lymphokine II thus induced and produced is
It can be easily purified and separated by a known purification and separation method, for example, salting out, dialysis, filtration, centrifugation, concentration, lyophilization, etc., and collected. When a higher degree of purification is required, for example, adsorption-elution on an ion exchanger, gel filtration and isoelectric focusing, electrophoresis, ion exchange chromatography, high performance liquid chromatography, column chromatography, affinity. By combining known methods such as chromatography, specific activity of about 10 ⁹ units /
It is also possible to collect new lymphokine II with the highest purity of mg protein.

In addition, the new lymphokine II thus obtained,
A warm-blooded animal other than human is immunized as an antigen, antibody-producing cells are collected from the animal, and the cells are fused with myeloma cells, and the resulting fused cells are fused cells having anti-new lymphokine II antibody-producing ability. The selected monoclonal cells were grown, and the produced monoclonal antibody was purified using, for example, an immobilized monoclonal antibody obtained by reacting with bromocyanine-activated sepharose to obtain highly pure new lymphokine II in high yield. Harvesting can also be used advantageously.

A new lymphokine II purified and produced in this way
Has a physicochemical property of molecular weight 20,000 ± 2,000 isoelectric point pI = 6.2 ± 0.3 mobility Disc-PAGE, R _f = 0.29 ± 0.02 UV absorption spectrum maximum absorption at around 280 nm Solubility in solvent, saline Or soluble in phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform Color reaction shows protein positive reaction by Lowry method or micro burette method, and sugar positive reaction by phenol sulfate method or anthrone sulfate method It exhibits cytotoxic activity against _L929 cells, and virtually no cytostatic activity or interferon activity against KB cells. Activity stability in aqueous solution Up to 60 ° C depending on the condition of keeping at pH7.2 for 30 minutes. Stable, 4
Stable within the range of pH 4.0 to 11.0 under the condition of keeping at ℃ for 16 hours It was found to be stable for more than 1 month when stored frozen at -10 ℃.

It was also found that the new lymphokine II has the ability to damage and kill not only mouse _L929 cells but also many human tumor cells, but does not substantially damage normal human cells. .

Therefore, the new lymphokine II, as a composition containing the same, a new lymphokine II-sensitive disease, for example, among the preventive and therapeutic agents for malignant tumors, conventionally, various malignant tumors of humans that have been extremely difficult to treat. It can be advantageously used as a therapeutic agent.

The activity of the new lymphokine II is as follows: KB cells as target cells,
_{Alternatively} , it was measured using _L929 cells. That is, when KB cells are used, the Canon Chemotherapy Report (Ca
ncer Chemotherapy Reports) Parts 3, Vol.
3, No.2, analogously as described in September 1972, to measure the growth inhibitory activity of KB cells, in the case of using L ₉₂₉ cells, E. Pick (E.Pick) ed, Tsumoa necrosis factor in Rinhokainzu (Tumor Necrosis Factor in
Lymphokines) Vol. II, pp. 245-249, Academic Press (1981), the cytotoxic activity given to _L929 cells in the presence of actinomycin D was measured. At this time, the activity when suppressing the growth of _L929 cells by 50% was _defined as 50 units. In the present specification, unless otherwise stated, the activity measurement method using _L929 cells was adopted.

The activity of interferon, which is highly species-specific for humans, is reported in "Protein Nucleic Acid Enzymes" Vol. 20, No. 6, pp. 616-643 (1975). It was measured by the half method.

The hemagglutination value is written by JESalk,
The Journal of Immunology
Immunology) Vol.49, page 87 (1944).

 Next, the present invention will be described experimentally.

Experiment A-1 Preparation of partially purified new lymphokine II A newborn hamster was injected with an antiserum prepared by a known method from a rabbit to weaken the immune response of the hamster, and then BALL-1 cells were transplanted subcutaneously into the hamster. Then, the animals were bred for 3 weeks in the usual manner. The subcutaneously formed tumor was excised, cut into small pieces, dispersed in physiological saline, and loosened. The obtained cells were washed with serum-containing RPMI 1640 medium (pH 7.2) and suspended in the same medium at about 2 × 10 ⁶ / ml. To this cell suspension, Sendai virus having a hemagglutination titer of about 400 per ml was added and kept at 37 ° C. for 24 hours to induce and produce new lymphokine II.

This was centrifuged at about 1,000 g at about 4 ° C. to remove the precipitate, and the resulting supernatant was dialyzed for 20 hours against physiological saline containing pH 7.2, 0.01M phosphate buffer, Furthermore, the filtrate obtained by microfiltration is passed through an antibody column on which anti-interferon antibody is immobilized, the non-adsorbed fraction is collected, and the active fraction is collected by the chromatofocusing method and concentrated. Then, it was freeze-dried to obtain a powder containing new lymphokine II activity.

The specific activity of this powder was about 10 ⁶ units / mg protein. The yield of new lymphokine II is about 1 per hamster.
It was 20 million units.

Experiment A-2 Preparation of anti-new lymphokine II antibody The new lymphokine II obtained by the method of Experiment A-1 was dissolved in physiological saline to a protein concentration of about 0.05 w / v%, and Freund's complete adjuvant emulsified. The same amount of the liquid is mixed, 0.2 ml of this mixed solution is subcutaneously injected into a mouse,
The mice were immunized by the same injection again 7 days later. The cells to antineoplastic lymphokines II antibody having antibody-producing ability allowed the induction generator, the spleen was excised from the mice, minced dispersed spleen cells and mouse myeloma cells P ₃ -X63-Ag8 "flow Laboratories obtained ( Flow Laboratories) "was prepared in serum-free Eagle's minimal basic medium at 50 W / V%
Polyethylene glycol-1000 solution (pH 7.2, temperature 37
At ⁴⁰ ° C / ml) and float for 5 minutes at 10 ⁴ / ml, and then dilute 20-fold with the basic medium. Then, Davison et al., Somatic Cell Genetics, Vol. .2, pages 175 to 176 (1976), fused cells that can grow in hypoxanthine-aminopterin-thymidine culture solution were collected, and the anti-new lymphokine II antibody-producing ability was collected from the fused cells. Fused cells were selected. About 10 ⁶ cells per mouse were transplanted into the abdominal cavity of the obtained fused cells, which were then bred for 2 weeks and then sacrificed to collect body fluids such as ascites and blood.
After centrifugation, the supernatant was salted out with ammonium sulfate to collect a precipitate fraction having a saturation degree of 30 to 50% and then dialyzed. Further, this liquid was used as a new lymphokine II obtained by the method of Experiment A-1 to bromocyane. Affinity chromatography was performed using immobilized new lymphokine II gel obtained by reacting with activated sepharose at room temperature to obtain an anti-new lymphokine II antibody fraction,
After dialysis, the mixture was concentrated and freeze-dried to collect a monoclonal antibody powder of new lymphokine II.

This product showed immunologically specific neutralizing activity against the cytotoxic activity of new lymphokine II.

Experiment A-3 Preparation of highly purified new lymphokine II and its physicochemical properties The partially purified product of new lymphokine II prepared by the method of Experiment A-1 was immobilized with the monoclonal antibody prepared by the method of Experiment A-2. The active fraction of the new lymphokine II was collected by affinity chromatography using the gelified gel, dialyzed, concentrated and lyophilized.

This product was a highly purified new lymphokine II with a specific activity of about 10 ⁹ units / mg protein.

 Using this product, physicochemical properties were investigated.

Molecular weight K Weber and M Osborne (K.We
ber and M. Osborn), Journal of Biological Chemistr
y), Vol.244, page 4406 (1969).
-Checked by polyacrylamide gel electrophoresis. That is, in the presence of 0.1% SDS, a sample of about 10 μg was loaded on a 10% acrylamide gel column, and after running for 4 hours at 8 mA per column, staining with Coomassie blue gave a single molecule with a molecular weight of 20,000 ± 2,000. A sharp strip was obtained. This single zone had a new lymphokine II activity.

Isoelectric point 25, using gel for isoelectric focusing, product name AMPHOLINE PAGPLATE (pH 3.5 to 9.5), manufactured by LKB, Sweden
As a result of electrophoresis for 2 hours with W, the isoelectric point pI was 6.2 ± 0.3.

Electric Mobility BJDavis, Annuals of the New York Academy of Sciences
s) According to the description in Vol.121, page 404 (1964), load about 10 μg of a sample onto a 7.5% acrylamide gel column and adjust the pH to 8.
3. After electrophoresing at 3 mA per column for 2 hours, extraction and electromobility determined by measuring the activity gave R _f 0.29 ± 0.02.

UV absorption spectrum Spectrophotometer made by Shimadzu Corporation, product name UV-250
As a result of investigating the absorption spectrum in the ultraviolet using 280n
It showed the maximum absorption around m.

Solubility in solvent Soluble in water, physiological saline or phosphate buffer It was sparingly soluble or insoluble in ethyl ether, ethyl acetate or chloroform.

Color reaction The protein positive reaction was shown by the Lowry method or the micro burette method, and the sugar positive reaction was shown by the phenol sulfate method or the anthron sulfate method.

Action It showed cytotoxic activity against _L929 cells. Virtually no cytostatic or interferon activity against KB cells was shown.

Activity stability in aqueous solution 1) Thermal stability A sample of about 1 × 10 ⁵ units / ml was kept at pH 7.2 for 30 minutes at each temperature, and the residual activity was measured. As a result, it was stable up to 60 ° C. .

2) pH stability 0.1 ml of a sample containing approximately 1 × 10 ⁶ units / ml was added to each pH buffer solution (pH 2 to 7
… Mcllvaine buffer, pH 7-
8 ... Phosphate buffer, pH 8-11 ...
Glycine-NaOH buffer
ffer)) Add 1 ml and hold at 4 ℃ for 16 hours.
As a result of adjusting the pH of the ml to pH 7.2 with 0.05M phosphate buffer and measuring the remaining activity, it was stable in the range of pH 4.0 to 11.0.

3) Stability about 1 × 10 ⁵ against DISPASE
Add dispase (protease derived from Bacillus bacterium produced by Godo Shusei Co., Ltd.) to 100 units / ml to 100 units / ml, react at pH 7.2, temperature 37 ° C for 0 to 2 hours, and sample economically And 1 w / v bovine serum albumin
The reaction was stopped by adding so as to become%. As a result of measuring the residual activity of new lymphokine II in this solution, new lymphokine II
Was unstable by dispase treatment, and the activity of new lymphokine II was lost along with the reaction.

Stability by freezing storage An aqueous solution of pH 7.2 was frozen at 10 ° C and stored for 1 month, then thawed and the activity was measured. As a result, no decrease in activity was observed.

From the above results, the new lymphokine II has physicochemical properties which have not been described in the literature, which is clearly different from the lymphokines such as lymphotoxin, TNF and interferon which are known in the art.

Experiment B-1 Growth inhibitory effect on malignant tumor cells The new lymphokine II obtained by the method of Experiment A-1 or Experiment A-3 was used to examine the growth inhibitory effect on various human-derived cells.

A new lymphokine II prepared by the method of Experiment A-1 or Experiment A-3 was prepared by culturing 1 day by culturing 10 ⁶ cells of various human origin in 1 ml of a known nutrient medium supplemented with fetal bovine serum. Saline containing 50 or 500 units of
0.1 ml was added and the mixture was cultured at 37 ° C for 2 days. After culturing, the Applied Microbiolog
y), Vol. 22, No. 4, pages 671 to 677 (1971), the live cells are stained with the stain neutral red, and then the stain is stained with acid ethanol. After elution, the amount of living cells was measured from the absorbance of the eluate at 540 nm.

In the control experiment, 0.1 ml of physiological saline containing no new lymphokine II was used.

 The cell growth inhibition rate (%) was calculated from the following formula.

The results are shown in Table 1.

As is clear from the results in Table 1, new lymphokines II
Was found to have little effect on normal cells and markedly suppress the growth of various malignant tumor cells. It was also found that the effect may be not only highly purified but partially purified.

Experiment B-2 BALB / C mice were transplanted with mouse sarcoma Meth-A cells,
From the 10th day after the transplantation, the new lymphokine II obtained by the method of Experiment A-3 was dissolved in physiological saline and
Intravenous injection was performed once at 100 or 1,000 units / Kg for 15 days. Then, the mice were sacrificed and the tumor weight was measured.

 The results are shown in Table 2.

Experiment B-3 BALB / C-derived nude mice were subcutaneously transplanted with human breast cancer tissue pieces, and the tumor volume became about 200 mm ³ ,
New lymphokine II obtained by the method of Experiment A-1 or Experiment A-3 was dissolved in physiological saline once a day,
Intravenous injection was performed at 100 or 1,000 units / Kg for 20 days.
Thereafter, the nude mice were sacrificed and the weight of the tumor was measured.

 The results are shown in Table 3.

Experiment B-4 BALB / C-derived nude mice were subjected to human breast cancer tissue slices Experiment B-
In the same manner as in No. ³ , the new lymphokine II and / or human α-interferon obtained by the method of Experiment A-3 from the time when the tumor volume became about 200 mm ³ was dissolved in a physiological saline and the daily dose was 1 Intravenous injection was performed once for 20 days.

Thereafter, the nude mice were sacrificed and the weight of the tumor was measured. In the control experiment, new lymphokine II and human α
-Saline without interferon was used.

 The results are shown in Table 4.

From the results shown in Table 4, the use of new lymphokine II in combination with interferon markedly enhances its antitumor effect.

Experiment B-5 An acute toxicity test of the new lymphokine II obtained by the method of Experiment A-3 was carried out using 20-day-old mice.
The toxicity of neolymphokine II was extremely low, and the LD ₅₀ when injected intraperitoneally was found to be 10 ⁹ units or more.

As is clear from the above experiments, the novel lymphokine II of the present invention is effective not only in vitro but also in vivo to suppress the growth of malignant tumors, and is stable in terms of its effective dose. Is extremely high.

The dose of the novel lymphokine II of the present invention per adult is 5
~ 500,000,000 units, preferably 5 to 10,000,000 units for topical injection and topical application such as eye drops, 10 to 50,00 for transdermal or transmucous application such as ointments or suppositories.
0,000 units, 50 to 10 for whole body injection such as intravenous injection and intramuscular injection
The dosage is 0,000,000 units and 500 to 500,000,000 units in the case of oral administration, but it can be increased or decreased depending on the usage or the symptoms. If necessary, it can be prepared into a pharmaceutical preparation by a conventional method together with any conventional pharmaceutical carrier, base or excipient. Considering the toxicity, effective amount and safety of the new lymphokine II, it is desirable to use 5 units or more of new lymphokine II per gram of pharmaceutical preparation.

The shape of the preventive or therapeutic agent for a new lymphokine II-sensitive disease containing the new lymphokine II can be freely selected according to its purpose.

For oral administration, enteric-coated preparations such as capsules, tablets, and powders, for rectal administration, rectal suppositories, and for injection, for example, freeze-dried injection that is dissolved in distilled water for injection before use. It can also be used as an agent, other nasal drops or eye drops, and an ointment.

Further, when treating a malignant tumor using a new lymphokine II, for example, by taking a part of the tumor of a patient and treating it with the new lymphokine II to enhance the immunogenicity of the tumor, the tumor patient The malignant tumor can be treated more effectively by returning it to the body. Further, along with the new lymphokine II various anti-tumor agents, for example interferon, TNF, lymphotoxin, other lymphokines such as TCGF, β-1,3 glucan, arabinomannan,
Lipopolysaccharide, OK-432 (Picibanil), PSK
(Crestin), antitumor polysaccharides such as lentinan, methotrexate (MTX), fluorouracil (5-FU)
It is also advantageous to further enhance the antitumor effect of the new lymphokine II by concomitantly using antimetabolites such as the above and antibiotics such as doxorubicin (ADM) and mitomycin C (MMC).

Hereinafter, an example of production of the new lymphokine II of the present invention will be described in Example A, and a new lymphokine II of the present invention will be described in Example B.
Production examples of various drugs which are the compositions of

Example A-1 Human-derived lymphoblastoid cells BALL-1 cells were inoculated into Eagle's minimal basal medium (pH 7.4) supplemented with 20% fetal bovine serum, and in vitro (in vitro) at 37 ° C. according to a conventional method. in vitro). The obtained cells were washed with Eagle's minimum basic medium (pH 7.4) containing no serum, and suspended in the same medium at about 1 × 10 ⁷ / ml. Approximately 1 ml of Sendai virus is added to this suspension.
1,000 hemagglutination titers were added and kept at 38 ° C for 1 day to induce and produce new lymphokine II. This was centrifuged at 4 ° C. and about 1,000 g, and the resulting supernatant was dialyzed for 15 hours against physiological saline containing pH 7.2, 0.01 M phosphate buffer, and further microfiltered to obtain a filtrate. Was applied to a column of anti-interferon antibody in the same manner as in Experiment A-1, and the non-adsorbed fraction thereof was purified by affinity chromatography using a gel column of a monoclonal antibody by the method described in Experiment A-3, concentrated, and then purified. A concentrate of new lymphokine II having an activity of about 10 ⁹ units / mg protein was obtained.

The active yield was about 1.5 million units per suspension at the time of induction production.

Example A-2 A neonatal hamster was injected with an antiserum prepared from a rabbit by a known method to weaken the immune response of the hamster, and then subcutaneously cultured into a human-derived lymphoblastoid cell BALL. -1 cells were transplanted and then reared by a usual method for 3 weeks. A tumor of about 15 g under the skin was excised, cut into small pieces,
It was dispersed in physiological saline and loosened. The obtained cells were washed with serum-free RPMI 1640 medium (pH 7.2) and suspended in the same medium at about 5 × 10 ⁶ / ml. To this suspension, about 1,000 hemagglutinin titers per ml and about 10 µg of endotoxin derived from E. coli were added per ml, and the mixture was kept at 37 ° C for 1 day to induce and produce new lymphokine II. About 4
Centrifugation at about 1,000 g at ℃, to remove the precipitate, the resulting supernatant was dialyzed for 21 hours against physiological saline containing pH 7.2, 0.01M phosphate buffer, and further microfiltered. The obtained filtrate was purified by using an antibody column in the same manner as in Example A-1, and the resulting solution was concentrated and freeze-dried to obtain a new lymphokine II powder having a specific activity of about 10 ⁹ units / mg protein. It was

 The activity yield was about 32 million units.

Example A-3 Human nude lymphoblastoid cells TALL-1 cells that had been cultured and cultured were transplanted into the abdominal cavity of grown nude mice, and then the mice were reared for 5 weeks by a conventional method. The Newcastle disease virus having a hemagglutination titre of about 3,000 was inactivated into the abdominal cavity with ultraviolet rays in advance and injected, and after 24 hours, the animals were slaughtered to collect ascites. Then, the powder was purified in the same manner as in Example A-2, concentrated and dried to obtain new lymphokine II powder.

The activity yield was about 3.5 million units per nude mouse.

Example A-4 Mature normal mice were pre-irradiated with about 400 rem of X-rays to weaken the immunocompetence of the mice, and then human-derived lymphoblastoid cells Mono-1 which had been cultured subcutaneously in the mice were cultured.
The cells were transplanted and then reared by a usual method for 3 weeks. A tumor mass of about 10 g subcutaneously was excised, and cells were dispersed in the same manner as in Example A-2. This cell was used in Example A-2.
After suspending in the same manner as above, approximately 500 hemagglutination titers of Sendai virus and ml of concanavalin A are added to this suspension.
0.8 μg was added and the mixture was kept at 37 ° C. for 1 day to induce and produce new lymphokine II. Thereafter, purification, concentration and drying were carried out in the same manner as in Example A-2 to obtain a new lymphokine II powder.

 The activity yield was 20 million units per mouse.

Example A-5 Human-derived lymphoblastoid cells Namalva cells cultured in the same manner as in Example A-2 were transplanted to a newborn hamster, and then the hamsters were reared for 4 weeks by a usual method. A mass of about 20 g subcutaneously was loosened in the same manner as in Example A-2 to about 3
A cell suspension of × 10 ⁶ / ml was obtained. To this suspension, about 1,000 hemagglutinating titers of Sendai virus were added and maintained at 36 ° C. for 2 days to induce and produce new lymphokine II, which was then purified and concentrated in the same manner as in Example A-1 to concentrate new lymphokine II. A liquid was obtained.

The activity yield was about 22 million units per hamster.

Example A-6 A human-derived lymphoblastoid cell NALL-cultured into a plastic cylindrical diffusion chamber having an internal volume of about 10 ml provided with a membrane filter having a pore size of 0.5 micron.
One cell was suspended in physiological saline and embedded in the abdominal cavity of the grown rat. After the rats were bred for 4 weeks in the usual way, the diffusion chamber was taken out. It was found that the concentration of human-derived cells thus obtained was about 5 × 10 ⁸ / ml, which was about 10 ² times or more higher than that when grown in an in vitro nutrient medium in a carbon dioxide incubator. . The cells were suspended in the same manner as in Example A-2, and Newcastle disease virus having a hemagglutination value of about 500 ml / ml was added thereto after being inactivated by ultraviolet rays, and phytohemagglutinin was added at about 50 μg / ml. Add 1 at 37 ℃
The new lymphokine II was induced and produced by keeping it for a day. After that,
The powder was purified in the same manner as in Example A-2, concentrated and dried to obtain new lymphokine II powder.

 The activity yield was about 8 million units per rat.

Example A-7 Human-derived cell line CCRF-CEM cells were transplanted into fertilized chicken eggs kept at 37 ° C. for 5 days, and then kept at 37 ° C. for 1 week. After breaking this egg, proliferating cells were collected. The cells were suspended at 5 × 10 ⁶ / ml in the same manner as in Example A-1. To this suspension, about 500 hemagglutinating titers per ml of Sendai virus were added and kept at 37 ° C. for 1 day to induce and produce new lymphokine II, which was then purified in the same manner as in Example A-2, concentrated and dried. A powder of new lymphokine II was obtained.

 The activity yield was about 700,000 units per 10 fertilized eggs.

Example B-1 Injection Solution New lymphokine II500 prepared by the method of Example A-2,
Dissolve 000 units in 200 ml physiological saline and filter aseptically using a membrane filter. Fill 2 ml each of the filtrate into a sterilized glass container, freeze-dry, and stopper this to give a freeze-dried powder formulation.

The product is suitable for treating breast cancer, lung cancer, liver cancer, leukemia and the like.

Example B-2 Injection In the production of the injection of Example B-1, α-derived from lymphoblastoid cells together with 500,000 units of new lymphokine II.
A freeze-dried powder preparation was obtained in the same manner as in Example B-1, except that 300,000,000 units of interferon were dissolved in 200 ml of physiological saline.

This product exerts a markedly superior therapeutic effect on various malignant tumors such as breast cancer, lung cancer, liver cancer, gastric cancer and leukemia, as compared with the injection of Example B-1.

Example B-3 Ointment The new lymphokine II prepared by the method of Example A-3 was ground to a small amount of liquid paraffin according to a conventional method, and then vaseline was added to give an ointment at 20,000 units / g.

The product is suitable for treating skin cancer, breast cancer, lymphoma and the like.

Example B-4 Eye Drop 800 ml of distilled water, 5 ml of β-phenylethyl alcohol and new lymphokine II 20,00 prepared by the method of Example A-4.
Add salt to make it isotonic with 0,000 units and 1,000 with distilled water
ml was used as eye drops.

 The product is suitable for treating retinoblastoma and the like.

Example B-5 Enteric coated tablet When the new lymphokine II prepared by the method of Example A-7 was tabletted using a mixture of starch and maltose according to a conventional method, the new lymphokine II was added per 1 tablet (100 mg) of the product. 20
A tablet was produced by incorporating it so that the amount would be 0,000 units, and methylcellulose phthalate was coated thereon to give an enteric coated tablet.

The product is suitable for treating colorectal cancer, colon cancer, liver cancer and the like.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1:91)

Claims (4)

[Claims] 1. A physicochemical property has a molecular weight of 20,000 ± 2,000, an isoelectric point pI = 6.2 ± 0.3, and a mobility Disc-PAGE, Rf = 0.29 ± 0.02. An ultraviolet absorption spectrum has a maximum absorption in the vicinity of 280 nm. Soluble in saline or phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Shows protein positive reaction by Lowry method or micro burette method, sugar by phenol sulfuric acid method or anthrone sulfuric acid method A cytoplasmic positive action L ₉₂₉ shows cytotoxic activity against ₉₂₉ cells, and virtually no cytostatic or interferon activity against KB cells.Activity stability in aqueous solution Stable up to 60 ℃, 4 ℃
Stable in the range of pH 4.0 to 11.0 by keeping for 16 hours at room temperature. New lymphokine II which is stable for more than 1 month when stored frozen at -10 ℃. 2. A process for producing a new lymphokine II, which comprises reacting an inducer with a human-derived cell having the following physicochemical properties and having the ability to produce a new lymphokine II to produce and collect the new lymphokine II. Molecular weight 20,000 ± 2,000 Isoelectric point pI = 6.2 ± 0.3 Rf = 0.29 ± 0.02 by mobility Disc-PAGE UV absorption spectrum Maximum absorption near 280 nm Solubility in solvent Water, physiological saline or phosphate buffer Soluble, sparingly soluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Positive protein reaction by Lowry method or micro burette method, positive sugar reaction by phenol-sulfuric acid method or anthrone-sulfuric acid method Action on _L929 cells It shows cytotoxic activity, but virtually no cytostatic or interferon activity on KB cells. Activity stability in aqueous solution Stable up to 60 ° C under conditions of pH7.23 for 30 minutes, 4 ° C
Stable in the range of pH 4.0 to 11.0 by keeping for 16 hours at -10 ° C Stable for more than 1 month when stored frozen at -10 ℃ 3. Physicochemical properties have a molecular weight of 20,000 ± 2,000 isoelectric point pH = 6.2 ± 0.3 Rf = 0.29 ± 0.02 in mobility Disc-PAGE, maximum absorption near UV absorption spectrum of 280 nm Solubility in solvent water, physiology Soluble in saline or phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Shows a protein-positive reaction by the Lowry method or the micro burette method, and sugar by the phenol-sulfuric acid method or anthrone-sulfuric acid method. A positive quality reaction L Cytotoxicity against _L929 cells and virtually no cytostatic or interferon activity against KB cells Activity stability in aqueous solution Stable up to 60 ℃, 4 ℃
Stable in the range of pH 4.0 to 11.0 under the condition of holding for 16 hours at a temperature of −10 ° C., a new lymphokine II, which is stable for more than 1 month when stored frozen, is purified using a monoclonal antibody showing specificity to A method for producing the new lymphokine II according to claim 2, which is characterized in that it is collected. 4. Physicochemical properties are: molecular weight 20,000 ± 2,000 isoelectric point pI = 6.2 ± 0.3 mobility Disc-PAGE, Rf = 0.29 ± 0.02 UV absorption spectrum maximum absorption near 280 nm Solubility in solvent water, physiology Soluble in saline or phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Shows protein positive reaction by Lowry method or micro burette method, sugar by phenol sulfuric acid method or anthrone sulfuric acid method Quality-positive reaction L Cytotoxicity against ₉₂₉ cells, virtually no cytostatic or interferon activity against KB cells, activity stability in aqueous solution Conditions to hold at pH 7.2 for 30 minutes Stable up to 60 ℃ by 4 ℃
An antitumor agent characterized by containing a new lymphokine II which is stable in the range of pH 4.0 to 11.0 under the condition of being kept for 16 hours at −10 ° C. and is stable for one month or more when stored frozen.