JPH0523755B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a new lymphokine having cytotoxic activity against tumor cells. As lymphokines that have cytotoxic activity against tumor cells, lymphotoxin derived from animals including humans, and tumoa necrosis factor (hereinafter referred to as "TNF") derived from animals other than humans are known. Lymphotoxins are co-authored by Ryuichi Aoki et al., “Lymphokines,” New Immunology Series 6, pp. 87-105 (1979)
Igaku Shoin, Bloom B.R.
R.) and Glade PR
Co-authored “In Vitro Methods in Cell Medication with Immunity”
Methods in Cell-Mediated Immunity”, Academic Press (1971)
and “Cellular Immunology”
Immunology), Vol. 38, pp. 388-402 (1978
The TNF is described in the Proceedings of the National Academy of the USA, such as Carswell EA.
Science of the USA), Volume 72, No. 9, No.
pp. 3666-3670 (1975) and E.
Pick) “Tsumoa Necrosis Factor”
In Lymphokines (Tumor Necrosis)
Factor in Lymphokines), Volume 235~
272 pages, Academic Press (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 that has undescribed physical and chemical properties that are completely different from those previously known lymphokines, established a method for producing it, and confirmed its cytotoxic activity against various malignant tumor cells, and found its uses. The present invention was completed by establishing the following. That is, the present invention has the following physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=6.2±0.3 Mobility Disc-PAGE, Rf=0.29±0.02 Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvent Water; Soluble in physiological saline or phosphate buffer Slightly 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 protein by the phenol sulfuric acid method or anthrone sulfuric acid method. Shows a quality positive reaction Action Shows cytotoxic activity against L ₉₂₉ cells and virtually no cytostatic activity or interferon activity against KB cells Activity stability in aqueous solution Conditions of holding at pH 7.2 for 30 minutes Stable up to 60℃, pH 4.0 to 4.0 by holding at 4℃ for 16 hours
Stable within the range of 11.0 The present invention relates to a method for producing a new lymphokine (this substance is referred to as a new lymphokine throughout this specification) that is stable for one month or more when stored frozen at -10°C. For example, the new lymphokine production method uses human-derived cells that have the ability to produce new lymphokines, e.g.
It may be produced by treating leukocytes, lymphocytes, cultured cells, etc. with an inducing agent. 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 transplanted directly into the body of a warm-blooded animal other than humans, or are inoculated into a diffusion chamber to reduce the supply of body fluids of the warm-blooded animal. 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 increases significantly. The cultured human-derived cells used in the present invention may be cells that can be transplanted into the body of a warm-blooded animal other than humans and easily proliferate, and that also have the ability to produce new lymphokines. nucleic acid enzyme
Various human-derived cell lines described in "Vol. 20, No. 6", pages 616-643 (1975) can be used. Among others, the Journal of Clinical Microbiology
Microbiology), Volume 1, pp. 116-117 (1975
Namalwa cells described in 2010), I. Miyoshi, ``Nature'',
BALL-1 cells, TALL-1 cells, and NALL-1 cells described in Vol. 267, pp. 843-844 (1977)
1 Cell, The Journal of Immunology, Volume 113, No.
M-7002 cells described on pages 1334-1345 (1974),
B-7101 cells, “Tissue Culture” Vol. 6, No. 13, 527
~ JBL cells, described on page 546 (1980),
EBV-Sa cells, EBV-wa cells, EBV-HO cells, MOLT-3 cells, other BALM-2 cells, CCRF-SB cells (ATCC CCL120) CCRF
- Established lymphoblastoid cell lines such as CEM cells and DND-41 cells, as well as normal mononuclear cells and granular white blood cells, were treated with various viruses, drugs, radiation, etc. to create culture lines. Cells and the like are suitable. In addition, the genes that have the ability to produce new lymphokines in these human-derived cells can be used, for example, by means of cell fusion using polyethylene glycol or Sendai virus, or by enzymes such as DNA ligase, restriction enzymes (nucleases), and DNA polymerases. They may be treated by genetic recombination to increase their proliferation rate or increase their ability to produce new lymphokines per cell, and are limited to the established cell lines described herein. It's not a thing. 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 grown, 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 capable of blocking the passage of animal cells without transplanting human-derived cells into the animal body, such as membrane filters ^{, ultrafiltration} membranes, or Diffusion chambers of various shapes and sizes known in the art, each equipped with a fluorophore or the like, are buried in the animal's body, for example, in the abdominal cavity, and the above-mentioned culture is carried out within the chamber while being supplied with body fluids containing nutrients from the animal's body. Any established human-derived cell line can be grown. In addition, if necessary, a chamber in which the solution containing nutrients in the chamber is connected to body fluids in the animal body and perfused is attached to the surface of the animal body, for example, to check the growth state 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 also 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. It is also possible to cause the new lymphokine inducer to act within the animal's body after it has grown. For example, a new lymphokine inducer is directly applied to human cells grown in suspension in ascites 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 removed from the body of a non-human animal and a new lymphokine inducing agent can be applied to induce the production of new lymphokines in vitro. For example, human-derived cells grown in ascites are collected, or tumors containing human-derived cells generated subcutaneously are excised and dispersed, and the resulting cells are placed in a nutrient medium kept at approximately 20 to 40 degrees Celsius to achieve a cell concentration. is 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 then 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, first, a new lymphokine is induced to be produced in the animal body using grown human-derived cells, and then human-derived cells collected from a specific part or the whole animal are added to the human-derived cells in the animal 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 individual animal 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. Also,
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 through these induced productions, but also human interferon, which is highly species-specific, is 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 performance liquid chromatography, column chromatography. By combining known methods such as affinity chromatography and affinity chromatography, the specific activity can be reduced to approx.
It is also possible to obtain the highest purity new lymphokines of ¹⁰⁹ units/mg protein. 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 produced in this way has the following physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=6.2±0.3 Mobility Rf=0.29±0.02 on Disc-PAGE Ultraviolet absorption spectrum Maximum absorption near 280 nm Solubility in solvents Soluble in water, physiological saline, or phosphate buffer Slightly 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 shows a positive carbohydrate reaction by the anthrone sulfate method Action: Shows cytotoxic activity against L ₉₂₉ cells, does not substantially show cytostatic activity against KB cells or interferon activity Activity stability in aqueous solution pH 7.2 Stable up to 60°C by holding at 4°C for 30 minutes; pH 4.0 to 4.0 by holding at 4°C for 16 hours
Stable in the range of 11.0 It was found that it is stable for more than 1 month when stored frozen at -10℃. It was also found that the new lymphokine has the ability to damage and kill not only mouse _L929 cells but also many 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
Reports) Parts 3, Volume 3, No. 2 (September 1972), measure the growth inhibitory activity of KB cells, and when using L ₉₂₉ cells,
Edited by E.Pick, Tumor Necrosis Factor in Lymphokines
Necrosis Factor in Lymphokines), Vol.
245-249, as described in Academic Press (1981).
Cytotoxic activity against _L929 cells in the presence of actinomycin D was measured. At this time, the activity when inhibiting the proliferation of _L929 cells by 50% was defined as 50 units. 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 FL 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. Hemagglutination titer is determined by JE
Salk), The Journal of Immunology, Volume 49,
It was measured according to the method described on page 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 formed under the skin was removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were added with serum.
The cells were washed with RPMI 1640 medium (pH 7.2) and suspended in the same medium at a density of approximately 2×10 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 pH 7.2 and 0.01M phosphate buffer. , the filtrate obtained by microfiltration is passed through an antibody column on which an anti-interferon antibody is immobilized, the non-adsorbed fraction is collected, and the active fraction is collected by chromatography, Concentration and lyophilization yielded a powder containing new lymphokine activity. The specific activity of this powder was approximately 10 ⁶ units/mg protein. Furthermore, the yield of the new lymphokine was approximately 20 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 mice, and 7 days later, the mice were immunized by the same injection again. Anti-neo-lymphokine antibodies were induced to be produced in cells capable of producing the antibody, and the spleen was removed from this mouse, and the resulting spleen cells and mouse myeloma cells P ₃ -X63-
Ag8 “Flow Laboratories (Flow
Laboratories) into a 50W/V% polyethylene glycol-1000 solution (pH 7.2, temperature 37°C) prepared with serum-free Eagle's minimal basic medium.
They were suspended at a concentration of 10 ⁴ /ml and kept for 5 minutes, then diluted 20 times with the above-mentioned basic medium.
Cell Genetics (Somatic Cell
Genetics), Vol. 2, pp. 175-176 (1976), fused cells that can proliferate in a hypoxanthine-aminobuterine-thymidine culture solution were collected, and the anti-neolymphokine was extracted from the fused cells. Fusion cells capable of producing antibodies were selected. Approximately 10 ⁶ of the obtained fused cells were transplanted into the peritoneal cavity of each mouse and kept for 2 weeks. After that, 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 to 50% was collected, then dialyzed, and this liquid was further used in Experiment A-1.
Affinity chromatography was performed using the immobilized new lymphokine gel obtained by reacting the new lymphokine obtained by the method with bromcyan-activated cepharose at room temperature to obtain an anti-new lymphokine antibody fraction, which was dialyzed and concentrated. The monoclonal antibody powder of the new lymphokine was collected by freeze-drying. 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 purified parts of new lymphokine prepared by the method of Experiment A-1 were 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. The physical and chemical properties of this product were investigated. Molecular Weight K.Weber and M.Osborn, Journal of Biological Chemistry
Biological Chemistry, Vol. 244, p. 4406 (1969), SDS-polyacrylamide gel electrophoresis was performed. i.e. 0.1%
Approximately 10 μg of the sample was loaded onto a 10% acrylamide gel column in the presence of SDS, and after electrophoresis at 8 mA per column for 4 hours, Coomassie blue was applied.
A single sharp band with a molecular weight of 20,000±2,000 was obtained. This single band had neolymphokine activity. Isoelectric point Gel for isoelectric focusing, manufactured by LKB, Sweden, product name: AMPHOLINE PAGPLATE
(pH 3.5 to 9.5) at 25 W for 2 hours, the isoelectric point pI was 6.2±0.3. Electrical Mobility BJDavis, Annuals of the New York Academy
of Sciences (Annuals of the New
Youk Academy of Sciences), Volume 121, No. 404
(1964), approximately 10 μg of the sample was loaded onto a 7.5% acrylamide gel column, electrophoresed for 2 hours at pH 8.3 and 3 mA per column, extracted, and the electrical mobility was determined from the activity measurement. However, Rf0.29±
It was 0.02. Ultraviolet absorption spectrum Spectrophotometer manufactured by Shimadzu Corporation, product name
The absorption spectrum in the ultraviolet region was examined using UV-250, and the maximum absorption was found around 280 nm. Solubility in solvents: Soluble in water, physiological saline, or phosphate buffer; 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. Effect: Showed cytotoxic activity against _L929 cells. KB
Substantially no cytostatic activity or interferon activity against cells was shown. Activity stability in aqueous solution 1 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. 2 pH stability 0.1 ml of a sample of approximately 1 x 10 ⁶ units/ml was added to each pH buffer (pH 2-7...Mcllvain buffer, pH 7-8...Phosphate buffer, pH 8-8). 11...Glycine-sodium hydroxide buffer (Glycine-sodium hydroxide buffer)
After adding 1 ml of NaOh buffer and keeping it at 4℃ for 16 hours, this 0.1 ml was adjusted to pH 7.2 with 0.05M phosphate buffer and the remaining activity was measured. It was stable in the range of 11.0 to 11.0. 3. Stability against DISPASE Add dispase (protease derived from Bacillus bacteria manufactured by Godo Shusei Co., Ltd.) to a sample of approximately 1 x 10 ⁵ units/ml to a concentration of 100 units/ml.
Bovine serum albumin was obtained by reacting for 0 to 2 hours at pH 7.2 and temperature of 37°C, and economically sampling.
The reaction was stopped by adding 1w/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 during frozen storage An aqueous solution at 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 has physical properties described at the end of the literature that are clearly different from previously known lymphokines such as lymphotoxin, 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 culturing, Applied Microbiology, No. 22
vol., No. 4, pp. 671-677 (1971), live cells were stained with the stain Neutral Red, and the stain was subsequently eluted with acid ethanol. The amount of viable cells was measured from the absorbance of the eluate at 540 nm. Note that 0.1 ml of physiological saline containing no new lymphokine was used in the control experiment. The cell proliferation inhibition rate (%) was calculated from the following formula. Cell growth inhibition (%) = (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 had little effect on normal cells, but was found 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 from 10 days after the transplant, 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] * At a risk rate of 5% or less, compared to the control value,
There is a statistically significant difference.
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 m
Experiment A- ¹ or Experiment A
- The new lymphokine obtained by method 3 is dissolved in physiological saline and administered at 100 or
Intravenous injection was given at 1000 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.

[Table] * At a risk rate of 5% or less, compared to the control value,
There is a statistically significant difference.
Experiment B-4 Human breast cancer tissue fragments were transplanted into BALB/C-derived nude mice in the same manner as in Experiment B-3, and from the time when the tumor volume reached approximately 200 ^mm3 , new lymphokines obtained by the method of Experiment A-3 were detected. and/or human α-interferon dissolved in physiological saline was intravenously injected once daily for 20 days. Thereafter, the nude mice were sacrificed and the weight of the tumor was measured. Note that in the control experiment, physiological saline containing no new lymphokine and human α-interferon was used. The results are shown in Table 4.

[Table] There is a statistically significant difference.
From the results in Table 4, the antitumor effect of the new lymphokine is significantly enhanced when used in combination with interferon. Experiment B-5 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 extremely high considering its effective dose. expensive. 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 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, it is desirable that the new lymphokine be used in an amount of 5 units or more per gram of the pharmaceutical preparation. The form of a new lymphokine-sensitive disease preventive or therapeutic agent containing a new lymphokine can be freely selected depending on the 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 a new lymphokine, for example, a part of a patient's tumor is taken and treated with the new lymphokine to increase the immunogenicity of the tumor, and then the tumor patient This malignant tumor can be treated more effectively by returning it to the body. In addition to new lymphokines, various antitumor agents such as interferon, TNF, lymphotoxin, other lymphokines such as TCGF, β-1,3 glucan, arabinomannan, lipopolysaccharide, OK-432, etc.
(Picibanil), PSK (Krestin), anti-tumor polysaccharides such as lentinan, antimetabolites such as methotrexate (MTX), fluorouracil (5-FU), antibiotics such as doxorubicin (ADM), mitomycin C (MMC), etc. It is also possible to advantageously use the new lymphokine in combination to further enhance the antitumor effect. Hereinafter, two to three embodiments of the present invention will be described. Example 1 Human-derived lymphoblastoid BALL-1 cells were inoculated into Eagle's minimal basal medium (pH 7.4) supplemented with 20% fetal bovine serum, and incubated in vitro at 37°C according to a conventional method. were cultured in suspension. The obtained cells were washed with serum-free Eagle's minimal basic medium (pH 7.4) and suspended in the same medium at a density of about 1×10 ⁷ /ml. Approximately per ml of Sendai virus is added to this suspension.
A hemagglutination titer of 1000 was added and kept at 38°C for 1 day to induce the production of new lymphokines. This is 4℃, approx.
Centrifuge at 1000g, and the resulting supernatant is diluted to pH 7.2.
15 in saline containing 0.01M phosphate buffer
Experiment A
-1, and the non-adsorbed fraction was purified by affinity chromatography using a monoclonal antibody gel column as described in Experiment A-3, concentrated and compared. A concentrated solution of a new lymphokine with an activity of approximately 10 ⁹ units/mg protein was obtained. The activity yield is approx. per suspension during induction production.
It was 1.5 million units. Example 2 After injecting antiserum prepared from rabbits by a known method into newborn hamsters to weaken the hamster's immune response, human-derived lymphoblastoid cells BALL-1 were cultured subcutaneously in the hamsters. transplant the cells,
Thereafter, the animals were raised in the usual manner for 3 weeks. 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 (pH 7.2) and suspended in the same medium at approximately 5×10 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 1, and the resulting solution is concentrated and lyophilized to have a specific activity of approximately ¹⁰⁹ units/mg protein. A new lymphokine powder was obtained. The activity yield was approximately 32 million units. Example 3 Adult nude mice were intraperitoneally transplanted with cultured human-derived lymphoblastoid cells TALL-1 cells, 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 2 to obtain a new lymphokine powder. The activity yield was approximately 3.5 million units per nude mouse. Example 4 Human-derived lymphoblastoid cell Mono-1 cells were cultured subcutaneously in an adult normal mouse by pre-irradiating it with approximately 400 rem of X-rays to weaken the immune system of the mouse. were transplanted and then reared for 3 weeks in the usual manner. After removing a subcutaneous tumor weighing approximately 10 g, cells were dispersed in the same manner as in Example 2. After suspending the cells in the same manner as in Example 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 suspension was kept at 37°C for 1 day. Lymphokines were induced to be produced. Thereafter, the product was purified, concentrated, and dried in the same manner as in Example 2 to obtain a new lymphokine powder. Activity yield was approximately 20 million units per mouse. Example 5 Human-derived lymphoblastoid cells cultured in newborn hamsters in the same manner as in Example 2
Transplant the Namalva cells and then use the usual method for 4
They were kept for a week. Approximately 20 g of tumor formed under the skin was dissected in the same manner as in Example 2 to obtain a cell suspension of approximately 3 x 10 ⁶ /ml. Sendai virus was added to this suspension at a hemagglutination value of approximately 1000 per ml and kept at 36°C for 2 days to induce the production of new lymphokines.
A concentrated solution of the new lymphokine was obtained by purification and concentration in the same manner as above. The activity yield was approximately 22 million units per hamster. Example 6 Cultured human-derived lymphoblastoid NALL-1 cells were suspended in physiological saline in a plastic cylindrical diffusion chamber with a capacity of approximately 10 ml and equipped with a membrane filter with a pore size of 0.5 microns. This was then implanted into 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 grown in an in vitro nutrient medium in a carbon dioxide incubator. The cells were suspended in the same manner as in Example 2, and to this suspension was added New Katus disease virus with a hemagglutination value of about 500 per ml, which had been inactivated in advance with ultraviolet light, and about 50 μg of phytohemagglutinin per ml. The cells were kept at 37°C for 1 day to induce the production of new lymphokines. From then on,
The product was purified in the same manner as in Example 2, concentrated and dried to obtain a new lymphokine powder. The activity yield was approximately 8 million units per rat. Example 7 After transplanting human-derived cell line CCRF-CEM cells 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 grown in the same manner as in Example 1.
It was suspended at ×10 ⁶ /ml. Approximately 500 per ml of this suspension
Add Sendai virus with hemagglutination titer and incubate at 37°C.
The mixture was kept for one day to induce the production of new lymphokine, and then purified in the same manner as in Example 2, concentrated and dried to obtain a powder of new lymphokine. The activity yield was approximately 700,000 units per 10 fertilized eggs.

Claims (1)

[Claims] 1 Physical and chemical properties include: Molecular weight 20000±2000 Isoelectric point pI=6.2±0.3 Mobility Rf=0.29±0.02 in Disc-PAGE Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvent Water , soluble in saline or phosphate buffer,
Poorly 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 carbohydrate reaction by the phenol sulfuric acid method or anthrone sulfuric acid method.Action against L ₉₂₉ cells Shows cytotoxic activity, does not substantially show cytostatic activity against KB cells, and interferon activity Stability of activity in aqueous solution Stable up to 60°C when kept at pH 7.2 for 30 minutes, kept at 4°C for 16 hours Depending on the conditions, the pH ranges from 4.0 to
Human-derived cells that are stable in the range of 11.0, have the ability to produce new lymphokines, are stable for more than 1 month when stored frozen at -10℃, and can be transplanted and proliferated into the body of a warm-blooded animal other than humans. A new lymphokine characterized in that a new lymphokine is produced from human-derived cells that are transplanted into the body of a warm-blooded animal other than humans and grown, or grown while being supplied with body fluids of a warm-blooded animal other than humans. Method for producing lymphokine. 2. The method for producing a new lymphokine according to claim 1, which comprises causing the proliferated human-derived cells to act with a new lymphokine inducing agent to produce the new lymphokine.