JPH0527640B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new lymphokine I having cytotoxic activity against tumor cells, a method for producing the same, and uses thereof. Lymphotoxins, tumoa necrosis factor, and the like are known as lymphokines that have cytotoxic activity against tumor cells. Lymphotoxins are co-authored by Ryuichi Aoki et al., "Lymphokines", New Immunology Series 6, pp. 87-105 (1979).
Co-edited by Igaku Shoin and Bloom BR & Glade PR “In
"Vitro methods in cell-mediated immunity"
Academic Press (1971) and Cellular
Immunology Vol. 38, pp. 388-402 (1978), etc., and the Tumoa necrosis factor is described in Carswe11 EA et al., Pr. Natl. Acad. Sci.,
USA, Vol.72, No.9 pp. 3666-3670 (1975)
and E.Pick ed. Tumor Necrosis Factor in
Lymphokines Vol.．． pp. 235-272, Academic
Press (1981), etc. Furthermore, recently, Haruo Onishi et al. have disclosed an antitumor glycoprotein, which is a type of lymphokine, in Japanese Patent Application Laid-Open No. 146293/1983. The inventors have been studying lymphokines for many years. As a result, we recognized the existence of a new lymphokine with physicochemical properties that are completely different from those previously known lymphokines, established a method for producing it, and also confirmed its cytotoxic activity against various malignant tumor cells, and established its use. The present invention was completed. That is, the present invention has the following physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=5.6±0.2 Mobility R _f =0.29±0.02 in Disc-PAGE Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvent Water , soluble in physiological saline or phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform Color reaction: Positive protein reaction by Lowry method or microbiuret method, phenol sulfuric acid method or anthrone sulfuric acid method Shows a positive carbohydrate reaction in KB cells.
Shows cytotoxic activity against L ₉₂₉ cells, with virtually no interferon activity Stability of activity in aqueous solution: Stable up to 60°C when kept at PH7.2 for 30 minutes; kept at 4°C for 16 hours By PH4.0
A new lymphokine that is stable in the range of 11.0 to 11.0 and unstable when treated with DISPASE and is stable for more than 1 month when stored frozen at -10°C (this substance is referred to as a new lymphokine throughout this specification), and its production Concerning methods and uses. The new lymphokine can be produced by causing an inducing agent to act on human-derived cells capable of producing the new lymphokine, such as leukocytes, lymphocytes, and cultured cells. Human-derived leukocytes and lymphocytes may be prepared by separating blood collected from humans. As the cultured human-derived cells, cells grown in vitro according to a conventional method can be used. However, in the case of the present invention, when growing cells in culture, they are directly transplanted into the body of a warm-blooded animal other than humans, or inoculated into a diffusion chamber, and the supply of body fluids of the warm-blooded animal is reduced. It is preferable to multiply while receiving. In other words, unlike in vitro cell proliferation, not only is there no need for, or can significantly save, nutrient media containing expensive serum, etc., but maintenance and management during cell proliferation is also extremely easy. Moreover, the new lymphokine activity produced by induction from the obtained cells is high. Methods using warm-blooded animals other than humans include transplanting cultured human-derived cells into the body of a warm-blooded animal other than humans, or creating a diffusion chamber that can receive the animal's body fluids. If the cells are implanted inside an animal and reared normally, the cells can easily proliferate using the nutrient-containing body fluids provided by the warm-blooded animal. Furthermore, compared to in vitro proliferation, the proliferation of these cells is stable, the proliferation rate is high, the amount of cells obtained is large, and the new lymphokine per cell is Another major feature is that the yield of I is significantly increased. The cultured human-derived cells used in the present invention may be cells that can be easily transplanted into the body of a warm-blooded animal other than humans and that have the ability to produce new lymphokines. Various human-derived cell lines described in "Protein Nucleic Acid Enzyme Vol. 20, No. 6", pp. 616-643 (1975) can be used. Among others, “Journal of Clinical
Namalva cells described in "Microbiology Vol. 1" pages 116-117 (1975), BALL-1 cells and TALL described in I. Miyoshi's "Nature Vol. 267" pages 843-844 (1977) -1 cell,
NALL-1 cells, “Journal of Immunology
Vol. 113” pages 1334-1345 (1974) M-
7002 cells, B-7101 cells, "Tissue Culture" Volume 6,
No. 13, pp. 527-546 (1980)
JBL cells, EBV-Sa cells, EBV-wa cells,
EBV-HO cells, MOLT-3 cells, and others
BALM2 cells, CCRF-SB cells (ATCC CCL
120) Cultured lymphoblastoid cell lines such as CCRF-CEM cells and DND-41 cells, as well as normal mononuclear cells and granular white blood cells, by treating them with various viruses, drugs, radiation, etc. Preferably, the cells are In addition, we can utilize genes that have the ability to produce new lymphokines in these human-derived cells by, for example, cell fusion methods using polyethylene glycol or Sendai virus, or enzymes such as DNA ligase, restriction enzymes (nucleases), and DNA polymerases. The cells may be treated with genetic recombination methods to increase their growth rate or increase their ability to produce new lymphokines per cell, and are limited to the established cell lines described herein. It's not something you can do. In the process of inducing the production of new lymphokines, which will be discussed later, these cells
They can be freely used alone or in combination of two or more. If necessary, leukocytes, lymphocytes, etc. collected and prepared from humans, for example, can be used in combination. The warm-blooded animals used in the present invention may be any animal in which human-derived cells can proliferate, such as birds such as chickens and pigeons, dogs, cats, monkeys, rabbits, goats, pigs, horses, cows, guinea pigs, Mammals such as rats, nude rats, hamsters, normal mice, and nude mice can be used. Transplanting human-derived cells into these animals may cause an unfavorable immune reaction, so in order to suppress such reactions as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or newborns. Preferably from childhood. In addition, these animals may be subjected to pretreatment such as irradiation with X-rays or gamma rays at a dose of about 200 to 600 rem, or injection of antiserum or immunosuppressants, etc., to weaken the immune response before transplantation. . If the animal used is a nude mouse or nude rat, the immune response is weak even if the animal is grown, so there is no need for these pretreatments, and cultured human-derived cells can be transplanted. , is particularly advantageous because it can be rapidly propagated. In addition, human-derived cells that have been cultured are first transplanted into hamsters, and then these cells are then transplanted into nude mice. It is also possible to further stabilize the proliferation of the derived cells and further increase the amount of new lymphokines induced and produced from them. In this case, transplantation can be done not only between the same species and the same genus, but also between the same class and phylum. The site within the animal body to which human-derived cells are transplanted may be any site where the transplanted cells can proliferate, such as the allantoic cavity, vein, abdominal cavity, or subcutaneous site. In addition, porous filtration membranes that can block the passage of animal cells without transplanting human-derived cells into the animal body, such as membrane filters and ultrafiltration membranes with pore diameters of approximately 10 ^-7 to ^{10 -5} m, are also available. Alternatively, a diffusion chamber of various shapes and sizes known in the art, equipped with a follow-up eye bar, etc., is buried in the animal's body, for example, in the abdominal cavity, and while receiving body fluids containing nutrients from the animal body, the above-mentioned Any cultured human-derived cells can be grown. If necessary, a chamber in which the solution containing nutrients in the chamber is connected to body fluids in the animal body for perfusion is attached to the surface of the animal body, for example, to check the state of transplantation of human-derived cells in the chamber. It is possible to see through the window provided on the surface of the chamber, and by making only this chamber part removable and replaceable, cells can be multiplied to the fullest of the animal's lifespan without having to be slaughtered, and the amount of cells produced per individual animal can be increased. It can also be increased further. These diffusion chamber-based methods do not allow direct contact between human-derived cells and animal cells;
Not only can only human-derived cells be easily collected, but there is also little risk of causing undesirable immune reactions, so there is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely. There is. The transplanted animal can be maintained and managed simply by continuing the normal care and management of the animal, and is convenient because no special handling is required even after transplantation. The purpose can usually be achieved in a period of 1 to 10 weeks for growing human-derived cells.
It has been found that the number of human-derived cells obtained in this manner reaches about 10 ⁷ to ^{10 12} or more per animal. In other words, the number of human-derived cells grown in an animal is approximately the same as the number of cells transplanted per animal.
10 ² to 10 ⁷ times, or even more, compared to about 10 ¹ to 10 ⁶ when grown in vitro in nutrient media.
twice or even more, making it extremely convenient for the production of new lymphokines. Any method can be used to induce and produce new lymphokines from human-derived cells grown in this manner. The new hokine-inducing agent can also act within the animal's body after it has proliferated. For example, a new lymphokine inducer is directly applied to human-derived cells transplanted in suspension in ascites fluid in the peritoneal cavity or tumor cells generated subcutaneously to induce the production of new lymphokines, and then the serum, ascites or New lymphokines can be purified and collected from tumors. Furthermore, proliferating cells derived from humans can be taken out from the body of a non-human animal and treated with a new lymphokine derivative in vitro to induce the production of new lymphokines. For example, human-derived cells grown in ascites are collected, or subcutaneous tumors containing human-derived cells are excised and dispersed, and the resulting cells are kept in a nutrient medium maintained at approximately 20 to 40°C. about
The cells may be suspended to a concentration of 10 ⁵ to ^{10 8} /ml, treated with a new lymphokine inducer to induce production of new lymphokines, and purified and collected. Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells can be left in the chamber or removed from the chamber and treated with a new lymphokine inducer to induce the production of new lymphokines. can. In addition, as mentioned above, human-derived cells obtained using warm-blooded animals other than humans can be further cultured in vitro for about 1 to 4 days to synchronize the cell proliferation generation, if necessary. It is also possible to induce the production of new lymphokines by applying a lymphokine inducer. In addition, for example, new lymphokines can be induced and produced in the animal's body by first proliferating human-derived cells, and then human-derived cells collected from a specific part or the whole of the same animal are injected into the animal's body. A method of inducing the production of new lymphokines, a method of using cells once used for the production of new lymphokines for induction production of new lymphokines more than once, or a method of implanting or replacing the chamber to be connected in the animal body. It is also possible to further increase the amount of new lymphokine produced per animal used by using methods such as increasing the number of cells obtained. The new lymphokine inducer of the present invention includes α
- Viruses, nucleic acids, nucleotides, etc. known as interferon inducers, phytohemagglutinin, concanavalin A, porkweed mitogen, lipopolysaccharide, endotoxin, polysaccharides, etc. known as γ-interferon inducers, Bacteria and the like are used as appropriate. For sensitized cells, antigens are also inducers of neolymphokines. Furthermore, when producing new lymphokines from human-derived cells, it is also possible to increase the production amount of new lymphokines by using α-interferon inducers and γ-interferon inducers together as new lymphokine inducers. It is. Furthermore, it has been found that not only new lymphokines are produced by these induced productions, but also human interferon, which is highly species-specific, is also produced at the same time. This makes it possible to simultaneously produce two or more valuable human physiologically active substances, and also to make advanced use of human-derived cells, which is extremely important in terms of supplying new lymphokines and human interferon in large quantities at low cost. It's convenient. The new lymphokine thus induced can be purified using known purification and separation methods such as salting out, dialysis,
It can be easily purified and separated and collected by filtration, centrifugation, concentration, freeze-drying, etc. If a higher level of purification is required, for example, adsorption to ion exchanger-elution, gel filtration and isoelectric point fractionation, electrophoresis, ion exchange chromatography, high speed liquid chromatography, column chromatography. By combining known methods such as graphie and affinity chromatography, it is possible to collect new lymphokines of the highest purity. In addition, the new lymphokine thus obtained can be used as an antigen to immunize warm-blooded animals other than humans.
Antibody-producing cells were collected from the animal, these cells were fused with myeloma cells, fused cells having the ability to produce anti-neolymphokine antibodies were selected from the resulting fused cells, and the selected cells were proliferated to produce It can also be advantageously used to purify a monoclonal antibody using, for example, an immobilized monoclonal antibody obtained by reacting with bromcyan-activated sepharose to collect a highly purified new lymphokine in high yield. The new lymphokine purified and manufactured in this way has the following physical and chemical properties: molecular weight 20000±2000, isoelectric point pI=5.6±0.2, mobility by Disc-PAGE, R _f =0.29±0.02, and ultraviolet absorption spectrum around 280 nm. Maximum absorption Solubility in solvents Soluble in water, physiological saline, or phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform Color reaction Shows a positive protein reaction by Lowry method or microbiuret method; Shows a positive carbohydrate reaction using the phenol sulfate method or anthrone sulfate method. Action: Has cell growth inhibitory activity against KB cells.
Shows cytotoxic activity against L ₉₂₉ cells and virtually no interferon activity Stability of activity in aqueous solution Up to 60°C by holding at PH7.2 for 30 minutes
Stable, PH4.0 when kept at 4℃ for 16 hours
Stable in the range of 11.0 to 11.0, unstable by DISPASE treatment It was found to be stable for more than 1 month when stored frozen at -10°C. It was also found that the new lymphokine has the ability to damage and kill not only mouse _L929 cells but also various human tumor cells, but has no substantial damage to normal human cells. Therefore, new lymphokines can be used as compositions containing them to treat new lymphokine-sensitive diseases, such as preventive and therapeutic agents for malignant tumors, as well as various human malignant tumors that have traditionally been considered extremely difficult to treat. It can be advantageously used as an agent. The activity of new lymphokines as target cells
It was measured using KB cells or _L929 cells. That is, when using KB cells, Cancer
Chemotherapy Reports Parts 3, Vol.3, No.
2. Measure the growth inhibitory activity of KB cells according to the description in September 1972, and when using L ₉₂₉ cells, measure the growth inhibitory activity of KB cells according to the description in E.Pick, Tumor Necrosis Factor in
“Lymphokines” Vol., pp.245-249,
Cytotoxic activity against _L929 cells in the presence of actinomycin D was measured according to the description in Academic Press (1981). In this specification, unless otherwise specified, an activity measurement method using _L929 cells was adopted. The activity of interferon, which is highly species-specific to humans, was determined using known human amnion-derived EL cells, as reported in "Protein Nucleic Acid Enzyme" Vol. 20, No. 6, pp. 616-643 (1975). It was measured by the plaque half-life method. The hemagglutination titer is determined by JESalk, “Journal of
It was measured according to the method of "Immunology" Vol. 49, p. 87 (1944). Next, the present invention will be explained through experiments. Experiment A-1 Preparation of partially purified new lymphokine Newborn hamsters were injected with antiserum prepared from rabbits using a known method to weaken the hamster's immune response, and BALL-1 cells were subcutaneously transplanted into the hamsters. Thereafter, the animals were raised in the usual manner for 3 weeks. The tumor that had formed under the skin was removed, cut into small pieces, and dispersed and loosened with physiological saline. The obtained cells were added with serum.
Wash with RPMI 1640 medium (PH7.2) and add approximately 2
It was suspended at ~10 ⁶ /ml. Sendai virus with a hemagglutination titer of about 400 per ml was added to this cell suspension and kept at 37°C for 24 hours to induce the production of new lymphokines. This was centrifuged at about 4°C and about 1000g to remove the precipitate, and the resulting supernatant was dialyzed for 20 hours against physiological saline containing PH7.2 and 0.01M phosphate buffer. The filtrate obtained by microfiltration was passed through an antibody column containing solidified anti-interferon antibodies, the non-adsorbed fraction was collected, and the active fraction was collected by chromatography. , concentrated and lyophilized to obtain a powder containing new lymphokine activity. The specific activity of this powder was approximately 10 ⁶ units/mg protein. Furthermore, the yield of new lihocain was approximately 30 million units per hamster. Experiment A-2 Preparation of anti-new lymphokine antibody The new lymphokine obtained by the method of Experiment A-1 was dissolved in physiological saline to a protein concentration of approximately 0.05 w/v%, and this was mixed with Freund's complete adjuvant emulsion. Mix equal amounts and make 0.2ml of this mixture.
was injected subcutaneously into the mouse, and 7 days later, the same injection was performed again to immunize the mouse. Spleen cells and mouse bone marrow cells P ₃ -X63- are obtained by inducing and producing anti-neolymphokine antibodies in cells that have the antibody-producing ability, removing the spleen from this mouse, and dispersing it into small pieces.
Ag8 (manufactured by Flow Laboratories) in a 50w/v% polyethylene glycol-1000 solution (PH7.2, temperature
After keeping the cells suspended at 10 ⁴ /ml for 5 minutes at 37°C, they were diluted 20 times with the above-mentioned basic medium.
Genetics, Vol. 2, pp. 175-176 (1976), collect fused cells that can grow in a hypoxanthine-aminopterin-thymidine culture solution, and use anti-neolymphokine antibodies from these fused cells. Fused cells with production ability were selected. Approximately 10 ⁶ of the obtained fused cells were transplanted into the peritoneal cavity of each mouse and raised for 2 weeks, after which the mice were sacrificed and body fluids such as ascites and blood were collected, centrifuged, and the supernatant was diluted with ammonium sulfate. The precipitate fraction with a saturation degree of 30-50% was collected and then dialyzed, and this solution was further treated with the new lymphokine obtained by the method of Experiment A-1 by reacting it with bromcyan-activated cepharose at room temperature. Affinity chromatography was performed using the immobilized new lymphokine gel to obtain an anti-new lymphokine antibody fraction, which was dialyzed, concentrated, and freeze-dried to collect the new lymphokine monoclonal antibody powder. This product showed immunologically specific neutralizing activity against the cytotoxic activity of new lymphokines. Experiment A-3 Preparation of highly purified new lymphokine and its physicochemical properties The partially purified product of new lymphokine prepared by the method of Experiment A-1 was immobilized with the monoclonal antibody prepared by the method of Experiment A-2. Affinity chromatography was performed using gel to collect the active fraction of the new lymphokine, which was dialyzed, concentrated, and lyophilized. This product is a highly purified new lymphokine, and its specific activity was approximately 10 ⁹ units/mg protein. Additionally, this product showed almost the same specific activity in an activity measurement method using KB cells. The physical and chemical properties of this product were investigated. Molecular weight K.Weber and M.Osborn, J.Biol.Chem.,
According to the description in Vol. 244, page 4406 (1969),
It was investigated by SDS-polyacrylamide gel electrophoresis. That is, approximately 10 grams of sample was loaded onto a 10% acrylamide gel column in the presence of 0.1% SDS,
After 4 hours of electrophoresis at 8 mA per column, it was extracted and the molecular weight was determined from the activity measurement.
It was ±2000. Isoelectric point Gel for isoelectric focusing, manufactured by LKB, Sweden, product name: AMPHOLINE PAGPLATE
As a result of electrophoresis at 25W for 2 hours using (PH3.5-9.5), the isoelectric point pI was 5.6±0.2. Electrical Mobility BJDavis, Ann.NYAcad.Sci., Vol.121,
Approximately 10 μg of the sample was loaded onto a 7.5% acrylamide gel column according to the description on page 404 (1964), and the PH
8.3. After 2 hours of electrophoresis at 3 mA per column, the electromobility was extracted and the activity was measured, and the R _f was 0.29±0.02. Ultraviolet absorption spectrum Spectrophotometer manufactured by Shimadzu Corporation, product name
As a result of examining the absorption spectrum in the ultraviolet region using UV-250, the maximum absorption was found around 280 nm. Solubility in solvents Soluble in water, saline or phosphate buffer. It was sparingly soluble to insoluble in ethyl ether, ethyl acetate, or chloroform. Color reaction A positive protein reaction was shown by the Lowry method or microbiuret method, and a positive carbohydrate reaction was shown by the phenol sulfuric acid method or anthrone sulfuric acid method. Action: Cytostatic activity against KB cells and
It showed cytotoxic activity against _L929 cells. Substantially no interferon activity was shown. Activity stability in aqueous solution (i) Thermal stability Approximately 1 × 10 ⁵ units/ml of sample at each temperature
After holding at pH 7.2 for 30 minutes, the remaining activity was measured and found to be stable up to 60°C. (ii) PH stability 0.1 ml of sample containing approximately 1×10 ⁶ units/ml was added to each PH buffer (PH2~7...Mcllvaine buffer.PH7~
8……Phosphate buffer.PH8～11……
Glycine-NaOH buffer) 1 ml and 4℃
After holding for 16 hours, this 0.1ml has a pH of 7.2,
As a result of adjusting the pH to 7.2 with 0.05M phosphate buffer and measuring the remaining activity, the pH ranged from 4.0 to 11.0.
It was stable within the range of . (iii) Stability against dispase (DISPASE) Dispase (protease derived from Bacillus bacteria manufactured by Godo Shusei Co., Ltd.) was added to a sample of approximately 1 × 10 ⁵ units/ml at a concentration of 100 units/ml, pH 7.2, The reaction was carried out at a temperature of 37° C. for 0 to 2 hours, samples were taken over time, and the reaction was stopped by adding bovine serum albumin to a concentration of 1 w/v%. As a result of measuring the residual activity of the new lymphokine in this solution, it was found that the new lymphokine was unstable due to dispase treatment, and the activity of the new lymphokine was lost as the reaction progressed. Stability due to frozen storage An aqueous solution of pH 7.2 was frozen at -10°C, stored for one month, thawed, and measured for activity. As a result, no decrease in activity was observed. From the above results, the new lymphokine is a previously known lymphotoxin,
It has physicochemical properties that are clearly different from lymphokines such as factor (TNF) and interferon. Experiment B-1 Growth inhibitory effect on malignant tumor cells Using the new lymphokine obtained by the method of Experiment A-1 or Experiment A-3, the growth inhibitory effect on various human-derived cells was investigated. Add 10 ⁶ cells of each type of human-derived cells to 1 ml of a known nutrient medium supplemented with fetal bovine serum, and after culturing for 1 day, add the new lymphokine prepared by the method of Experiment A-1 or Experiment A-3. 50 units or
0.1 ml of physiological saline containing 500 units was added and cultured at 37°C for 2 days. After incubation, Applied
Microbiology Vol. 22, No. 4, pp. 671-677 (1971
Live cells were stained with the stain Neutral Red according to the method described in 2010), the stain was then eluted with acid ethanol, and the eluate was
The amount of living cells was measured from the absorbance at 540 nm. In addition, 0.1 ml of physiological saline containing no new lymphokine was used in the control experiment. The cell growth inhibition rate (%) was calculated from the following formula. Cell growth inhibition rate (%) = (1-absorbance using new lymphokine/absorbance of control) x 100 The results are shown in Table 1.

[Table] As is clear from the results in Table 1, the new lymphokine was found to have almost no effect on normal cells, but to significantly suppress the proliferation of various malignant tumor cells. Furthermore, it has been found that the effect can be achieved not only by highly purified products but also by partially purified products. Experiment B-2 Mouse sarcoma Meth-A cells were transplanted into BALB/C mice, and then from the 10th day of transplantation, Experiment A-3
The new lymphokine obtained by the above method was dissolved in physiological saline and intravenously injected once daily at 100 or 1000 units/kg for 15 days. Thereafter, mice were sacrificed and tumor weights were measured. The results are shown in Table 2.

[Table] * There is a statistically significant difference when compared to the control value at a risk rate of 5% or less.
Experiment B-3 Human breast cancer tissue fragments were subcutaneously transplanted into the back of BALB/C-derived nude mice, and the tumor volume was approximately 200 mm ³
Experiment A-1 or Experiment A-
The new lymphokine obtained by method 3 is dissolved in physiological saline and administered at 100 or 1000 doses once daily.
Intravenous injections were given in units/Kg for 20 days. after that,
Nude mice were sacrificed and tumor weights were measured. The results are shown in Table 3.

[Table] * There is a statistically significant difference when compared to the control value at a risk rate of 5% or less.
Experiment B-4 An acute toxicity test of the new lymphokine obtained by the method of Experiment A-3 was conducted using mice 20 days after birth, and the toxicity of the new lymphokine was extremely low, and the LD when injected intraperitoneally. ₅₀ turned out to be more than 10 ⁹ units. As is clear from the above experiments, the new lymphokine of the present invention is effective in suppressing the growth of malignant tumors not only in vitro but also in vivo, and its stability is poor considering its effective dose. Extremely high. The daily dose for adults of the new lymphokine of the present invention is 5 to 500000000 units, preferably the dose for local application such as local injection and eye drops is 5 to 500000000 units.
10000000 units, 10 to 50000000 units for transdermal or transmucosal applications such as ointments or suppositories, 50 to 100000000 units for systemic injections such as intravenous and intramuscular injections, and 500 to 500000000 units for oral administration. The dosage can be increased or decreased as appropriate depending on usage or symptoms. If necessary, it can be prepared into a medical 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, it is desirable that the amount used is 5 or more units of the new lymphokine per gram of the medical preparation. The form of a new lymphokine-sensitive disease preventive or therapeutic agent containing a new lymphokine can be freely selected according to its purpose. Orally administered preparations include enteric preparations such as capsules, tablets, and powders; intrarectally administered preparations include rectal suppositories; and injections include, for example, freeze-dried injections that are dissolved in distilled water for injection before use. It can also be used as a nasal or eye drop, or as an ointment. In addition, when treating a malignant tumor using the new linkahoin, for example, a part of the patient's tumor is taken and treated with the new linkahoin to increase the immunogenicity of the tumor, and then the tumor patient is treated with the new linkahoin. This malignant tumor can be treated more effectively by returning it to the body. Hereinafter, Example A will describe a production example of the new lymphokine of the present invention, and Example B will describe a production example of various drugs that are compositions of the new lymphokine of the present invention. Example A-1 Human-derived lymphoblastoid BALL-1 cells were inoculated into Eagle's minimum basic medium (PH7.4) supplemented with 20% fetal bovine serum, and incubated in vitro ( in
suspension culture in vitro). The obtained cells were washed with serum-free Eagle's minimal basal medium (PH7.4),
The cells were suspended in the same medium at a concentration of approximately 1×10 ⁷ /ml.
Approximately 1000 hemagglutinating titers of Sendai virus per ml were added to this suspension, and the mixture was kept at 38°C for one day to induce the production of new lymphokines. About 1000g of this at 4℃
The resulting supernatant was dialyzed for 15 hours against physiological saline containing 0.01M phosphate buffer at pH 7.2, and the resulting filtrate was microfiltered in the same manner as in Experiment A-1. The non-adsorbed fraction was purified by affinity chromatography using a monoclonal antibody gel column as described in Experiment A-3 and concentrated to a specific activity of approximately ^109. A concentrated solution of new lymphokine with unit/mg protein was obtained. The activity yield is approx. per suspension during induction production.
It was 2 million units. Example A-2 Antiserum prepared from rabbits by a known method was injected into newborn hamsters to weaken the immune response of the hamsters, and then human-derived lymphoblastoid cells BALL were cultured subcutaneously. -1 cells were transplanted and then reared for 3 weeks in the usual manner. A subcutaneous tumor weighing approximately 15 g was removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were incubated with serum-free
The cells were washed with RPMI 1640 medium (PH7.2) and suspended in the same medium at a concentration of about 5 to 10 ⁶ /ml. Sendai virus was added to this suspension with a hemagglutination titer of approximately 1000 per ml and E.
Approximately 10 μg/ml of endotoxin derived from E. coli was added and kept at 37° C. for 1 day to induce the production of new lymphokines. This was centrifuged at about 4°C and about 1000g to remove the precipitate, and the resulting supernatant was
21 in saline containing 0.01M phosphate buffer
The filtrate obtained by time dialysis and further microfiltration is purified using an antibody column in the same manner as in Example A-1, and the resulting solution is concentrated and lyophilized to have a specific activity of approximately 10 ⁹
A new lymphokine powder with unit/mg protein was obtained. The activity yield was about 40 million units. Example A-3 Cultured human-derived lymphoblastoid cells TALL-1 cells were intraperitoneally transplanted into adult nude mice, and then raised in a conventional manner for 5 weeks. New Katsuru disease virus with a hemagglutination value of approximately 3000 was inactivated by ultraviolet rays and then injected into the abdominal cavity of the animal. After 24 hours, the animal was sacrificed and the ascites fluid was collected.
Thereafter, the product was purified and concentrated and dried in the same manner as in Example A-2 to obtain a new lymphokine powder. The activity yield was approximately 4 million units per nude mouse. Example A-4 A grown normal mouse was pre-irradiated with approximately 400 rem of X-rays to weaken the mouse's immune capacity, and then human-derived lymphoblastoid cells (Mono-) were cultured subcutaneously in the mouse. One cell was transplanted, and then raised for 3 weeks in the usual manner. After removing a subcutaneous tumor weighing approximately 10 g, the cells were dispersed in the same manner as in Example A-2. After suspending the cells in the same manner as in Example A-2, approximately 500 hemagglutination titer per ml of Sendai virus and 0.8 μg per ml of concanavalin A were added to this suspension, and the mixture was kept at 37°C for 1 day. and induced the production of new lymphokines. Thereafter, the product was purified, concentrated, and dried in the same manner as in Example A-2 to obtain a new lymphokine powder. Activity yield was 24 million units per mouse. Example A-5 Human-derived lymphoblastoid cells cultured in newborn hamsters in the same manner as in Example A-2
Transplant the Namalva cells and then use the usual method for 4
They were kept for a week. A subcutaneous tumor weighing about 20 g was loosened in the same manner as in Example A-2 to obtain a cell suspension of about 3 x 10 ⁶ /ml. Add ml of Sendai virus to Hongen suspension.
Approximately 1,000 red blood cell agglutination titer was added to the mixture and kept at 36° C. for 2 days to induce the production of new lymphokine. The mixture was then purified and concentrated in the same manner as in Example A-1 to obtain a concentrated solution of new lymphokine. The activity yield was approximately 26 million units per hamster. Example A-6 Cultured human-derived lymphoblastoid NALL-1 cells were placed in a plastic cylindrical diffusion chamber with a capacity of approximately 10 ml equipped with a membrane filter with a pore size of 0.5 microns in physiological saline. It was suspended and buried in the abdominal cavity of an adult rat. After the rats were housed in the usual manner for 4 weeks, the diffusion chambers were removed. The concentration of human-derived cells obtained in this way was approximately 5×10 ⁸ /
ml, which was found to be approximately 10 ² times more than when transplanted into an in vitro nutrient medium in a carbon dioxide incubator. This cell was used in Example A-
Suspend in the same manner as in step 2, and add about 500 mL per ml to this suspension.
The New Katsurus disease virus with hemagglutination titer was previously inactivated with ultraviolet rays and then approximately 50 μg/ml of phytohemagglutinin was added and kept at 37° C. for 1 day to induce the production of new lymphokines. Thereafter, it was purified, concentrated and dried in the same manner as in Example A-2 to obtain a new lymphokine powder. The activity yield was approximately 10 million units per rat. Example A-7 After transplanting human-derived CCRF-CEM cell line into fertilized chicken eggs kept at 37°C for 5 days,
It was kept at 37°C for one week. After the eggs were broken, proliferating cells were collected. These cells were suspended at 5×10 ⁶ /ml in the same manner as in Example A-1. Sendai virus with a hemagglutination titer of about 500 per ml was added to this suspension,
It was kept at 37°C for 1 day to induce the production of new linkine, and then purified and concentrated in the same manner as in Example A-2.
After drying, a new lymphokine powder was obtained. The activity yield was approximately 800,000 units per 10 fertilized eggs. Example B-1 Injection New lymphokine prepared by the method of Example A-2
Dissolve 1,500,000 units in 200 ml of physiological saline and filter aseptically using a membrane filter. Fill 2 ml of the filtrate into sterilized glass containers, freeze-dry them, and seal them tightly to obtain a freeze-dried powder preparation. This product is suitable for the treatment of breast cancer, lung cancer, liver cancer, leukemia, etc. Example B-2 Ointment The new lymphokine prepared by the method of Example A-3 was dissolved in a small amount of liquid paraffin according to a conventional method, and then vaseline was added to prepare an ointment containing 20,000 units/g. This product is suitable for treating skin cancer, breast cancer, lymphoma, etc. Example B-3 Eye Drops 800 ml of distilled water, 5 ml of β-phenylethyl alcohol, and the new lymphokine prepared by the method of Example A-4 were mixed with salt to make it isotonic to 2,000,000 units, and the volume was made up to 1,000 ml with distilled water. It was used as a drug. This product is suitable for the treatment of retinoblastoma, etc. Example B-4 Enteric-coated tablet When tabletting using a mixture of starch and maltose according to a conventional method, the new lymphokine prepared by the method of Example A-7 was added per tablet (100 mg) of the product.
Tablets were prepared by containing 200,000 units, and the tablets were coated with methyl cellulose phthalate to make enteric-coated tablets. This product is suitable for the treatment of colorectal cancer, colon cancer, liver cancer, etc.

Claims (1)

[Claims] 1 Physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=5.6±0.2 Mobility Rf=0.29±0.02 in Disc-PAGE Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvent Water , soluble in physiological saline or phosphate buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform Color reaction: Positive protein reaction by Lowry method or microbiuret method, phenol sulfuric acid method or anthrone sulfuric acid method Shows a positive carbohydrate reaction in KB cells.
Shows cytotoxic activity against L ₉₂₉ cells, with virtually no interferon activity Stability of activity in aqueous solution: Stable up to 60°C when kept at PH7.2 for 30 minutes; kept at 4°C for 16 hours By PH4.0
Stable in the range of 11.0 to 11.0, unstable by DISPASE treatment A new lymphokine that is stable for more than 1 month when stored frozen at -10°C. 2. A method for producing a new lymphokine, which comprises producing and collecting a new lymphokine by causing an inducing agent to act on human-derived cells having the ability to produce a new lymphokine having the following physical and chemical properties. Molecular weight 20000±2000 Isoelectric point pI=5.6±0.2 Mobility Rf=0.29±0.02 in Disc-PAGE Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvents Can be used in water, physiological saline or phosphate buffer Poorly soluble or insoluble in soluble, ethyl ether, ethyl acetate, or chloroform Color reaction Shows a positive protein reaction by the Lowry method or microbiuret method, and shows a positive carbohydrate reaction by the phenol sulfuric acid method or anthrone sulfuric acid method Action Against KB cells and cell proliferation inhibitory activity.
Shows cytotoxic activity against L ₉₂₉ cells, with virtually no interferon activity Stability of activity in aqueous solution: Stable up to 60°C when kept at PH7.2 for 30 minutes; kept at 4°C for 16 hours By PH4.0
Stable in the range of 11.0 to 11.0, unstable by DISPASE treatment Stable for more than 1 month when stored frozen at -10℃ 3 Physical and chemical properties: Molecular weight 20000±2000 Isoelectric point PH=5.6±0.2 Mobility Disc− PAGE, Rf = 0.29±0.02 Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvents Soluble in water, physiological 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 microbiuret method, and a carbohydrate-positive reaction by the phenol sulfuric acid method or anthrone sulfuric acid method.Action: Has cell proliferation inhibitory activity against KB cells.
Shows cytotoxic activity against L ₉₂₉ cells, with virtually no interferon activity Stability of activity in aqueous solution: Stable up to 60°C when kept at PH7.2 for 30 minutes; kept at 4°C for 16 hours By PH4.0
A new lymphokine that is stable in the range of 11.0 to 11.0, unstable by DISPASE treatment and stable for more than 1 month when stored frozen at -10°C, is purified using a monoclonal antibody specific for this lymphokine and collected. A method for producing a new lymphokine according to claim 2, characterized in that: 4 Physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=5.6±0.2 Mobility Disc-PAGE, Rf=0.29±0.02 Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvents Water, physiological saline or phosphorus Soluble in acid salt buffer, sparingly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform Color reaction: Shows a positive protein reaction by the Lowry method or microbiuret method, and shows a positive reaction for carbohydrates by the phenol sulfuric acid method or anthrone sulfuric acid method. Effect: It has cytostatic activity against KB cells.
Shows cytotoxic activity against L ₉₂₉ cells, with virtually no interferon activity Stability of activity in aqueous solution: Stable up to 60°C when kept at PH7.2 for 30 minutes; kept at 4°C for 16 hours By PH4.0
An antitumor agent characterized in that it contains a new lymphokine I that is stable in the range of 11.0 to 11.0 and unstable when treated with DISPASE and is stable for one month or more when stored frozen at -10°C.