JPS61115027A - Novel lymphokine ii, production and use thereof - Google Patents

Abstract

NEW MATERIAL:A lymphokine II (molecular weight, 20,000+ or -2,000; isoelectric point, pI=6.2+ or -0.3; mobility, Rf=0.29+ or -0.02 by Disc-PAGE; solubility, soluble in water, saline water and phosphate buffer solution, hardly soluble or insoluble in ethylether, etc.; color reactions, positive to protein by Lawry reaction, etc., positive to glucide by phenol sulfuric acid method, etc.; stability, stable at 60 deg.C for 30min at 7.2pH, stable at 4.0-11.0pH at 4 deg.C for 16hr, and stable for >=1 month in frozen state at -10 deg.C). USE:Agent for suppressing the proliferation of malignant tumor cells. PREPARATION:The novel lymphokine II is produced by treating a human- originated cell capable of producing the novel lymphokine II (e.g. leukocyte, lymphocyte, established cell, etc.) with an inducing agent. The limphokine is essentially harmless to the normal human cell.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel anti-inflammatory drug/I having cytotoxic activity against tumor cells, and its production method and uses.

Lymphokines having cytotoxic activity against tumor cells include lymphotoxy/ and Tumoa necrosis factor.

Lymphotoxy/ is co-authored by Takashi Aoki et al.
New Immunology Series 6.87-105 (1979) Igaku Shoin, Bloom B, Re & Glade P, R,
Co-edited rInVitro methods in cell
l mediated immunity
demic Press (1971) and Cel
lularImmunologyVow, 8B, 88
It is described in pages 8-402 (197F!N-), etc., and the Tsumoa Necrosis Factor is described in Carswell E.
R.A., et al., Pr., Natl.
Acad, Sct, +.

U, S, A,, Vol, 72, No, 98666~
3670 pages (1975) and E, Pickii T.
umor Necrosis Factor inLy
mphokines Vol, n, 235-27
2, Academic Press (1981), etc.

Furthermore, recently, Haruo Onishi et al. have disclosed an antitumor glycoprotein, which is a type of lymphocytosis, in Japanese Patent Application Laid-open No. 146298/1983.

The present inventors have been researching su/hokai/ for many years. As a result, we recognized the existence of a new type of lymphoid that has physicochemical properties that are completely different from those previously known, established a method for its production, and confirmed that it has cytotoxic activity against various malignant tumor cells. We established its use and completed the present invention.

That is, the physical and chemical properties of the present invention are: (1) Molecular weight: 20,000 + 2,000 (2) Isoelectric point pI = 6.2±0.3 (4) Mobility in Disc-PAGE, Rf = 0.29±0. 02■
Ultraviolet absorption spectrum Maximum absorption near 280 nm■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffers Slightly soluble to insoluble in ethyl ether, ethyl acetate, or chloroporm■ Color reaction Lowry method or microburet 0 effect showing a positive protein reaction by the phenol-sulfuric acid method or a carbohydrate-positive reaction by the phenol-sulfuric acid method or the anotrone-sulfuric acid method; 2. Shows cytotoxic activity against cells, and virtually no cytostatic activity against KB cells or interferon activity ■ Activity stability in aqueous solution: Stable up to 60'C when kept at pH 7.2 for 30 minutes, 4 Stable in the pH range of 4.0 to 11.0 when kept at ℃ for 16 hours. This invention relates to Hokai/X (this substance is referred to as new lymphokine I throughout this specification), its production method, and uses.

New Sunhokine/■ can be produced by causing an inducer to act on human-derived cells capable of producing New Sunhokine (2), such as leukocytes, lymphocytes, cultured cells, etc.

Human-derived white blood cells, 77 cells, 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 inoculated into a diffusion chamber/bar to absorb the body fluids of the warm-blooded animal. It is preferable to multiply while receiving supplies.

That is, unlike in vitro cell growth, not only is there no need for, or can significantly save on, nutrient media containing expensive serum, etc., but maintenance and management during cell growth is also extremely easy. In addition, it has the characteristic that the new phosphorus/II activity induced and produced from the obtained cells is high.

A method using warm-blooded animals other than humans involves transplanting cultured human-derived cells into the body of a warm-blooded animal other than humans.
Alternatively, if a diffusion chamber/par that can receive the animal's body fluids is buried in the animal's body and reared normally, the diffusion chamber/par that can receive the animal's body fluids can be used for normal rearing. Cells can easily proliferate.

Furthermore, compared to the case of in vitro, ro proliferation, the proliferation of these cells is stable, the proliferation rate is high, the amount of cells obtained is large, and furthermore, the number of cells per cell is Another major feature is that the yield of new lymphokine ■ is significantly increased.

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 have the ability to produce new/hokai/I. , for example, “Protein Nucleic Acid Enzyme Vol. 1.20, No. 6
Various human-derived cell lines described in J. J. 616-643 (1975) can be used. Among others, r Jou'rnal of C11nical
Microbiology Vol, I J116~
Namalv described on page 117 (1975)
a-cell, I, Miyoshi, “Natur@Vo
1.267 J pages 843-844 (1977) BALL-1 cells, TALL-1 cells, N
ALL-1 cells, “The Journal of Im
Munology Vol, 113 J 1384~
M-7002 cells described on page 1345 (1974), B
-7101 cells, “Tissue Culture” Vol. 6, No. 13, 52
7-546 (1980) K described JBL cells, EBV-8a cells, EBV-wa cells, EB
V-) IO cells, MOLT-3 cells, and other BALM
-2 cells, CCR'F-SB cells (ATCCccr.

120) Established lipoblastoid cell lines such as CCRF-CEM cells and DND-41 cells, as well as normal mononuclear cells and granular leukocytes, are treated with various viruses, drugs,
Cells treated with radiation or the like and cultured are suitable.

In addition, the genes having the new/anal production ability of these human-derived cells can be transferred by means of cell fusion using, for example, polyethylene/gelcoal or 7/daivirus.
DNAIJ gauze, restriction enzyme (nuclease), DNA
It may be treated by genetic recombination means using enzymes such as polymerases to increase its growth rate or increase its ability to produce new/hokai/]I per cell. It is not limited to the established cell lines described. These cells can be freely used alone or in combination of two or more types in the process up to the induced production of new/hokine 1, which will be described later. If necessary, leukocytes, lymphocytes, etc. collected and prepared from humans, for example, can be used in combination.

The warm-blooded animal used in the present invention may be any animal in which human-derived cells can proliferate, such as chickens, birds such as horses, dogs, cats, monkeys, rabbits, goats, pigs, horses, etc.
Salivary mammals such as cows, guinea pigs, rats, nude rats, hamster H 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.

Friend, you can pre-treat these animals by irradiating them with 200 to 600 rem of Enx-rays or gamma rays, or injecting them with antiserum or immunosuppressants, etc. to weaken their immune response and transplant them. good.

If the animal used is a nude mouse or nude rat, the immune response will be weak even if the animal is fully grown.
No pre-treatment was required.

This is particularly advantageous because cultured human-derived cells can be transplanted and rapidly proliferated.

Please note that it is not possible to transplant cultured human-derived cells into warm-blooded animals other than humans, such as first transplanting them into hamsters and growing them, and then transplanting these cells into nude mice. It is also possible to further stabilize the proliferation of human-derived cells and to increase the amount of new cells induced and produced from them.

In this case, transplantation may be performed 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, without transplanting human-derived cells into the animal body,
A porous filtration membrane capable of blocking the passage of animal cells, such as a membrane filter having a pore size of approximately 10 to 10 m.
1. Diffusion chambers of various known shapes and sizes equipped with ultrafiltration membrane casings or follower fibers are buried in the animal's body, for example in the abdominal cavity, and are supplied with body fluids containing nutrients from the animal body. , any of the human-derived cells cultured as described above can be grown in the chamber/bar.

In addition, if necessary, a chamber/bar that connects and perfuses the solution containing nutrients in the animal's body with the body fluid in the animal's body is attached to the surface of the animal's body, and the solution containing nutrients contained in the chamber/bar is It is possible to see the state of cell proliferation through a window provided on the surface of the animal, and it is also possible to make only a portion of the chamber removable and replaceable, allowing the cells to proliferate to the fullest lifespan without slaughtering the animal. It is also possible to further increase the amount of cells produced per animal.

Since human-derived cells do not come into direct contact with animal cells, methods using these diffusion chambers not only allow for easy collection of only human-derived cells, but also reduce the risk of causing an immune reaction. Therefore, there is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely.

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 period for growing human-derived cells is usually 1 to 10
The goal can be achieved within a week. The number of human-derived cells obtained in this way is approximately 10 to 1 per animal.
It was also found that the number reached 0 or more.

In other words, the number of human-derived cells grown in an animal body reaches approximately 10 to 10 times the number of cells transplanted per individual animal, or more, and is grown by inoculating into a nutrient medium outside the body. This is about 10 to 10 times higher than that of the previous model, or even more, which is extremely convenient for the production of new lymphokines.

Any method can be used to induce and produce Shin/Hokai/■ from the human-derived cells grown in this manner.

It is also possible to cause a new lymphokine-inducing agent to act in the animal's body where it has grown. For example, by directly acting on human-derived cells that have grown in suspension in ascites in the peritoneal cavity or on tumor cells that have arisen under the skin, inducing the production of new/hocain ■, Next, fresh Noho Kaino (2) may be purified and collected from the serum, ascites, or tumor.

It is also possible to remove human-derived proliferating cells from the body of a non-human animal and induce generation of new Nohokai 7I in vitro by applying an inducing agent.

For example, human-derived cells proliferated in ascites are collected, or subcutaneous tumors containing human-derived cells are excised and dispersed, and the resulting cells are placed in a nutrient medium maintained at approximately 20 to 40°C to achieve a cell concentration. It is sufficient to float it so that it is about 105 to 108/punishment, and to induce the production of new/hokai/■ by acting on it with an inducing agent, and to purify and collect it.

Furthermore, when human-derived cells are grown in a diffusion chamber/bar, the grown cells can be left in the chamber or removed from the chamber and treated with a new lymphokine inducer. It is also possible to induce generation of Nhokai/1.

In addition, as mentioned above, human-derived cells obtained using warm-blooded animals other than humans may be further cultured for 1 to 4 days in vitro to synchronize the cell proliferation generation, if necessary. After that, it is also possible to induce the production of Shinsunhokai 7K by applying an inducing agent.

In addition, for example, first, proliferated human-derived cells are induced to produce new hokines in the animal body, and then human-derived cells collected from a specific part or whole of the same animal are used. A method for inducing the production of new lymphokine ■ outside an animal body, and a method for using cells once used for the induction production of new lymphokine two or more times for the induction production of new lymphokine ■.

Alternatively, the amount of new lymphokine produced per individual animal can be further increased by increasing the number of cells that can be obtained by replacing the chamber/bar that is implanted or connected within the animal body.

New invention of the present invention/Hokai/I [Inducers include viruses, nucleic acids, nucleotides, etc. known as α-interferon inducers, phytohemagglutinin, concanavalin A1 porkweed mitose, etc. known as γ-interferon inducers Gen, lipopolysaccharide,
Polysaccharides, bacteria, etc. are used as appropriate.

Furthermore, for sensitized cells, antigens are also inducers of new/hokai/I.

Furthermore, when inducing the production of new/hokine ■ from human-derived cells, it is possible to induce the production of new hokine by using an α-interferon inducer and an r-interferon inducer together as the new/hokai/■ inducer. They are also free to increase production.

Furthermore, it has been found that not only Shin/Hokai/1 is produced by these induced productions, but also human interfero/, which is highly species-specific, is simultaneously produced.

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, and to supply new hokines and human interferon in large quantities at low cost. This is extremely convenient.

The thus-induced and produced new sulfuric acid/■ can be easily purified and separated by performing known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and freeze-drying. be able to. If a higher level of purification is required, for example, adsorption-elution on an ion exchanger,
By combining known methods such as gel filtration, isoelectric focusing, electrophoresis, ion-exchange chromatography, high-speed liquid chromatography, column chromatography, and affinity chromatography, the highest purity new/hocaine can be collected. It is also possible to do so.

In addition, a warm-blooded animal other than humans is immunized with the thus obtained Shinsunhokai/■ as an antigen, antibody-producing cells are collected from the animal, and these cells are fused with myeloma cells, From the obtained fused cells, fused cells having the ability to produce anti-lymphocytic/■ antibodies were selected.

The selected cells are grown and the produced monoclonal antibodies are reacted with, for example, Bromsia/activated Sepharose.
It can also be advantageously used to purify the resulting immobilized monoclonal antibody and collect highly pure Shin/Hokai/■ in high yield.

The new lymphokine ■ purified and manufactured in this way is
Physical and chemical properties: ■Molecular weight 20,000±2.000 ■Isoelectric point pI=6.2±0.8 ■Mobility Disc-PAGE: Rf”0.29 0.02■
Ultraviolet absorption spectrum Maximum absorption near 280 nm ■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer ■ Slightly soluble to insoluble in ethyl ether, ethyl acetate, or chloroform ■ Color reaction Lowry method or microviny Shows a positive protein reaction by the Rett method, and shows a positive carbohydrate reaction by the phenol-sulfuric acid method or the anotrone sulfuric acid method.Substantially shows no cell proliferation inhibitory activity or interferoactivity.Activity stability in aqueous solution: 30 at pH 7.2 It was found that it is stable up to 60°C when kept for 16 minutes, and stable in the pH range of 4.0 to 11.0 when kept at 4°C for 16 hours.It was found to be stable for more than 1 month when stored frozen at -10°C.

Also, new/hokai/l is mouse L, 2. Although it has the ability to damage and kill not only cells but also many human tumor cells, it has also been found that it does not substantially damage normal human cells.

Therefore, new/hokai/l [is new/hokai/! as a composition containing it, etc. [susceptible diseases, e.g.
Among preventive and therapeutic agents for malignant tumors, it can be advantageously used as a therapeutic agent for various human malignant tumors, which have been considered extremely difficult to treat.

The activity of new/hokine ■ is determined by KB cells as target cells,
or L9□, measured using cells. That is, when using KB cells, Cancer Chemothe
rapy Reports Part a, vol.
3, Teeth, 2, September 1972, when measuring the growth suppressive activity of KB cells and using IL929 cells, E. Pick, ed., Tunor Ne.
crosisFactor in ” Lymphok
ines' Vol, I, PP-245~249, A
2. Glue in the presence of actinomycete/D as described in Academic Press (1981); The cytotoxic activity exerted on cells was measured. In this specification, unless otherwise specified, L, 2. An activity measurement method using cells was adopted.

The activity of interferon, which is highly species-specific to humans, is described in “Protein Nucleic Acid Enzyme J Vol, 20, No. 6.616-6.
It was measured by the known Freak half-life method using human amnion-derived FL cells as reported on page 43 (1975).

The hemagglutination titer is determined by J, E, 5alk, The Jo.
Urnal of Immunology J Vol.
The measurement was carried out according to the method of P. 49.87, (1944).

Next, the present invention will be explained by experiment.

Experiment A-1 Preparation of partially purified new lymphokine (■) Inject neonatal hamsters with antiserum prepared from rabbits using a known method to weaken the hamster's immune response, and then transplant BALL-1 cells subcutaneously. After that, they 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 placed in RPMI 1640 medium (pH 7,2) supplemented with serum.
The cells were washed with water and suspended in the same medium at a density of about 2 x 10'/d. Sendai virus with a hemagglutination titer of about 400 per d was added to this cell suspension, and the suspension was kept at 37°C for 24 hours to induce the production of new lymphokine I.

This was centrifuged at about 4°C and about 1,000F to remove the precipitate, and the resulting supernatant was added to pH 7,2,0,01M IJ
The filtrate obtained by dialysis with physiological saline containing phosphate buffer for 20 hours and further microfiltration was
The non-adsorbed fraction is collected by applying the antibody to an immobilized antibody column, and then the active fraction is collected by chromatofocusing, concentrated, and lyophilized to renew the immunophokine activity. A powder containing was obtained.

The specific activity of this powder was approximately 106 units/η protein.

Furthermore, the yield of new lymphokine 1 was approximately 20 million units per hamster.

Experiment A-2 Preparation of anti-refractory lymphocytic 7Ti antibody Experiment A-1
Dissolve the new/hocain Wataru obtained by the method in physiological saline to a protein concentration of approximately 0.05 w/v%, mix this with equal amounts of Freund's complete adjuvant emulsion, and prepare this mixed solution. 0.2 m was injected subcutaneously into the mouse, and 7 days later, the same injection was repeated to immunize the mouse. Spleen cells and mouse myeloma cells P8-X68-Ag were obtained by inducing anti-fold/hokine I antibodies to be produced in cells capable of producing the antibody, removing the spleen from the mouse, and dispersing it into small pieces.
8 (manufactured by Flow Laboratories) and 5 prepared in serum-free Eagle's minimal basal medium.
0W/V% polyethylene glycol-1000 solution (p
H7.2, temperature 37°C), suspended at a concentration of 104/1 nl and kept for 5 minutes, diluted 20 times with the above basal medium, and then Dayλson etc.
ticCell Genetics, Vol. 2
．． According to the method reported on pages 175-176 (1976), fused cells that can proliferate in a hypoxanthine-aminopterin-thymidine culture solution were collected, and from these fused cells, fused cells having the ability to produce anti-lymphokine antibodies were obtained. selected. The obtained fused cells were intraperitoneally administered at a rate of about 1 per mouse.
After transplanting 0 and raising it for 2 weeks, it was sacrificed and ascites,
Body fluids such as blood are collected, centrifuged, the supernatant is salted out with ammonium sulfate, a precipitate fraction with a saturation level of 30-50% is collected, and then dialyzed.

Furthermore, this solution was subjected to affinity chromatography using immobilized Shinsunhokai/■ gel obtained by reacting Shinsunhokai 7II obtained by the method of Experiment A-1 with bromocyan-activated Sepharost at room temperature. An anti-lymphokine ■ antibody fraction was obtained, dialyzed, concentrated, and freeze-dried to collect a new lymphokine ■ monoclonal antibody powder.

This product showed immunologically specific neutralizing activity against the cytotoxic activity of Shin/Hokine I.

Experiment A-8 Preparation of highly purified Shin/Hokai 7T1 and its physicochemical properties A partially purified product of new lymphokine ■ prepared by the method of Experiment A-1 was used as a monoclonal antibody prepared by the method of Experiment A-2. Abfinity chromatography was performed using a gel immobilized with the active fraction of the new lymphokine ■, and
It was dialyzed, concentrated, freeze-dried, and dried.

This product is a highly purified new lymphokine 1 with a specific activity of approximately 109 units/η protein. Using this product, its physicochemical properties were investigated.

■For molecular weight, Weber and M, 0sborn, J, B
iol, Chem.

It was investigated by 5DS-polyacrylamide gel electrophoresis according to the description in Vol. 244, p. 4406 (1969). That is, in the presence of 0.1% SDS, approximately 10μ2 of the sample was loaded onto a 10 thiacrylamide gel column, and after electrophoresis at 8mA per column for 4 hours, it was extracted, and the molecular weight was determined from the activity measurement, which was 20,000±2. .000
Met.

■Isoelectric focusing gel 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 and 0.3.

■ Electrical mobility B, J, Davis 1Ann, N, Y, Aca
d, Sci,, Vol. 121.404 (196
Approximately 10 μg of the sample was loaded onto a 7.5 thiacrylamide gel column, pI (8,8), and electrophoresed for 2 hours at 3 mA per column, extracted, and the electrical mobility was determined from the activity measurement. When I calculated it, it was R, 0.29 soil, 0.02.

■ Ultraviolet absorption spectrum Spectrophotometer manufactured by Shimadzu Corporation, product name UV-2
As a result of examining the absorption spectrum in the ultraviolet region using 50,
Maximum absorption was observed around 280 nm.

(2) Solubility in solvents Soluble in water, physiological saline or phosphate buffer Ethyl ether and ethyl acetate fc were sparingly soluble or insoluble in chloroform.

■ A positive protein reaction was shown by the color reaction Lowry method or microburet method, and a positive carbohydrate reaction was shown by the phenol sulfuric acid method or the anotron sulfuric acid method.

■Effect, 2. It showed cytotoxic activity against cells. Substantially no cytostatic activity or interferon activity against KL cells was shown.

■ Activity stability in aqueous solution 1) Thermal stability approximately 1 x 10 units/m7! Samples were adjusted to pH 7,
2 for 30 minutes, the remaining activity was measured and found to be stable up to 60°C.

2) 0.1 d of sample with P)I stability of approximately I
, pH7~B-Phosphat buffer
, pH 8-11-Glycine-NaOHbu
After adding to 1 ml of Q and holding at 4°C for 16 hours, 1 ml of this Q was added to pH 7,2,0,05M IJ
/Results of adjusting the p)I to 7.2 with an acid salt buffer and measuring the remaining activity.

It was stable in the pH range of 4.0 to 11.0.

3) Stability against DISPASE: Approximately 1 x 105 units/a of sample was injected with dispase (protease derived from Pacillus bacteria manufactured by Godo Shusei Co., Ltd.) 1
00 units/-, pH 7.2, temperature 37
React for 0 to 2 hours at
The reaction was stopped by adding bovine serum Alpumi/ to a concentration of 1 w/v%.

As a result of measuring the residual activity of new/hokai/■ in this liquid,
Shin/Hokai 7N [was unstable due to disbase treatment, and the activity of Shin Shinhokai/II was lost as the reaction progressed.

(2) Stability due to frozen storage An aqueous solution with a pH of 7.2 was frozen at -10°C and stored for one month, then thawed and the activity was measured. As a result, no decrease in activity was observed.

From the above results, the new lymphokine/I has clearly different physicochemical properties from the previously known lymphokines such as lymphokine 7, TNF, and inter7ero7.

Experiment B-1 Growth inhibitory effect on malignant tumor cells Experiment A
-1, or new/hocaine obtained by the method of Experiment A-3■
was used to investigate the growth-inhibitory effect on various human-derived cells.

10 cells of each type of human-derived cells were placed in a known nutrient medium 1ral supplemented with fetal bovine serum, cultured for one day, and then added to the i.i. cells prepared by the method of Experiment A-1 or Experiment A-8.
9.1 ml of physiological saline containing 50 units or 500 units of IJ Inhokaino■ was added and cultured at 37°C for 2 days. % Applied Microbiolo after completion of culture
gy Vol, 22. No. 4.671-677 (
Living cells were stained with the stain Neutral Red according to the method described in (1971), and then the stain was eluted with acid ethanol, and the amount of viable cells was measured from the absorbance of the eluate at 540 nm. .

In addition, for the control experiment, 0.1 M of physiological saline not containing new nohocaine was used.

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

Cell growth inhibition rate (%) = The results are shown in Table 1.

Table 1 (Note) The numbers in the table indicate the cell proliferation inhibition rate (%).

As is clear from the results in Table 1, New/Hocain ■ is 1. It was found that it had little effect on normal cells, but significantly suppressed the proliferation of various malignant tumor cells. It has also 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 Math A cells were transplanted into BALB/C mice, and from day 10 after the transplant, new lymphokine I obtained by the method of Experiment A-3 was dissolved in physiological saline. Once daily, 100 or 1,000 units/Kf
Intravenous injections were given for 15 days. Thereafter, mice were sacrificed and tumor weights were measured.

The results are shown in Table 2.

Table 2 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 from the time when the tumor volume reached approximately 200 mm, Experiments A-1 and C were conducted using the method of Experiment A-3. The Shin/Hokaino ■ obtained above was dissolved in physiological saline and intravenously injected once daily at 100 or 1,000 units/KIi for 20 days. Thereafter, nude mice were sacrificed and tumor weights were measured.

The results are shown in Table 3.

“With a risk rate of 5% or less, there is a statistically significant difference compared to the control value.

Experiment B-4 An acute toxicity test of Shin/Hokaino 1 obtained by the method of Experiment A-8 was conducted using 20-day-old mice.
The toxicity of Shin/Hokai 7II was found to be extremely low, with a D5o of 109 units or more when injected intraperitoneally.

As is clear from the above experiments, the novelty/hokai/■ of the present invention is effective not only in vitro but also in vitro.
It is also effective in inhibiting the growth of malignant tumors in vivo.

Its stability is extremely high considering its effective dose.

The daily dose for adults of the new/hokai/■ of the present invention is 5 to 5.
500,000.000 units, preferably the locally applied dose such as local injection and eye drops is 5-10.000.
000 units, 10 to 50,000,000 units for transdermal or transmucosal applications such as ointments or suppositories, and 50 to 100,000,000 units for systemic injections such as intravenous and intramuscular injections.
unit, 500-500,000.000 for oral administration
Although it is a unit, it can be increased or decreased as appropriate depending on the usage or symptoms. Optional, conventional pharmaceutical carriers as required;
It can be prepared into a pharmaceutical preparation by a conventional method together with a base or excipient. The amount used is New Sun Hokai 7
Considering the toxicity, effective amount and safety of fi, it is desirable to contain 5 or more units of new lymphokine I per duram of pharmaceutical preparation.

The form of the new phosphokine 7T1t-containing preventive or therapeutic agent for sensitizing diseases can be freely selected depending on the purpose.

Oral preparations include enteric preparations such as capsules, tablets, and powders; intrarectal preparations include rectal suppositories; and injections include lyophilized injections that are dissolved in distilled water for injection. It can also be used as a nasal or eye drop, or as an ointment.

In addition, when treating a malignant tumor using Shinsunhokai/■, for example, a part of the patient's tumor is removed and this is replaced with
Treatment of this malignant tumor can be made more effective by increasing the immunogenicity of the tumor by treating it with hocaine (III) and then returning it to the tumor patient's body. In addition to Shinsunhokai/II, various antitumor agents such as interferon, TNF, lymphotoxin, TCGF, other glues/hocaines, β-1,3 gluca/, antitumor polysaccharides such as lipopolysaccharide, 5- It can also be advantageously used in combination with anti-metabolites such as F'U, antibiotics such as mitomycin, etc. to further enhance the antitumor effect of the drug.

Hereinafter, Example A will describe a manufacturing example of the new lymphokine (2) of the present invention, and Example B will describe manufacturing examples of various drugs that are the new lymphokine/Wataru compositions of the present invention.

Example A-1 Human-derived lymphoblastoid BALL-1 cells were cultured in Eagle's minimal basal medium (pH
7, 4) and in vitro (in vitro) according to the standard method at 3γC.
The cells were cultured in suspension in vitro. The obtained cells were washed with serum-free Eagle's minimal basal medium (pH 7.4) and suspended in the same medium to a concentration of approximately I x 107/mA'. Sendai virus was added per ml to this suspension. Add about 1.000 red blood cell agglutination value and keep it at 38℃ for 1 day to make it new/Hocain! Induce a friend. This is heated to 4℃, about 1,
00 (centrifuged at l, p the resulting supernatant) 17.2
The filtrate obtained by dialysis with physiological saline containing 0.01 M IJ y salt buffer for 15 hours and further microfiltration was applied to an anti-inotorferro/antibody column in the same manner as in Experiment A-1, and the The adsorbed fraction was purified by affinity chromatography using a monoclonal antibody gel column as described in Experiment A-8, and concentrated to a specific activity of approximately 109.
A concentrated solution of Shin/Hokai 7I having unit/η protein was obtained.

The activity yield was approximately 1.5 million units per lt of suspension during induction production.

Example A-2 Neonatal hamsters were injected with antiserum prepared from rabbits by a known method to weaken the immune response of the hamsters, and then cultured subcutaneously into human-derived lymphoblastoid cells. BALL-1 cells were transplanted and then reared for 3 weeks in the usual manner. A subcutaneous tumor of about 15 f was removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were placed in serum-free RPMI 1640 medium (pH 7,2).
The cells were washed with water and suspended in the same medium at a density of about 5 x 10 cells. To this suspension, approximately 1,000 hemagglutination titer per Sendai virus fag and E. coli-derived todoxin were added.
Approximately 10 μt per liter was added and kept at 37° C. for 1 day to induce the production of new lymphokine. This is about 4℃, about 1,
Centrifuge at 000 t to remove the precipitate.

The obtained supernatant was dialyzed for 21 hours against physiological saline containing p)I7.2.0.01 MIJ salt buffer, and the filtrate obtained by further microfiltration was treated in the same manner as in Example A-1. The resulting solution was concentrated and lyophilized to obtain a powder of the new lymphokine (2) having a specific activity of approximately 109 units/(2) protein.

The activity yield was approximately 32 million units.

Example A-3 Adult nude mice were intraperitoneally transplanted with cultured human-derived lymphoblastoid cells, TALL-1 cells, and then reared for 5 weeks in a conventional manner. Into this abdominal cavity, about a,oo
o Hemagglutination titer of Niecastle's disease virus was inactivated in advance by ultraviolet rays and injected, and 24 hours later, the animals were sacrificed and 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 powder of Shinsunhokai/■.

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

Example A-4 After pre-irradiating an adult normal mouse with approximately 400 rem of X-rays to weaken the immune capacity of the mouse, human-derived lymphoblastoid cells Mono were cultured subcutaneously in the mouse.
-1 cells were transplanted and then reared for 8 weeks in the usual manner. After removing a subcutaneous tumor of about 10F, Example A
Cells were dispersed in the same manner as in -2.

After suspending the cells in the same manner as in Example A-2, Sendai virus was added to the suspension at a hemagglutination value of about 500 per mt and conocanavalin A at 0.8 μt per d.
The mixture was kept at 37°C for 1 day to induce the production of new/hocain. Thereafter, the product was purified, concentrated, and dried in the same manner as in Example A-2 to obtain a powder of new lymphokine 1.

The activity yield was 40 million units per mouse.Example A-5 Neonatal hamsters were transplanted with human-derived lymphoblastoid Namalva cells cultured in the same manner as in Example A-2. Thereafter, they were raised in the usual manner for 4 weeks.

Approximately 201 subcutaneous tumors were loosened in the same manner as in Example A-2 to obtain a cell suspension of approximately 8 x 10'/III/. Approximately 1,000 ml of Sendai virus is added to this suspension.
0 red blood cell agglutination titer was added and the mixture was kept at 86°C for 2 days to induce the production of new hokain yI, and then purified and concentrated in the same manner as in Example A-1 to obtain a concentrated solution of new hokain I.

The activity yield was approximately 22 million units per musk.

Example A-6 Cultured human-derived lymphoblastoid cells NA in a 10 ml plastic cylindrical diffusion chamber/bar equipped with a metsublan filter with a pore size of 0.5 microns.
LL-1 cells were suspended in physiological saline and implanted into the peritoneal 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 thus obtained was approximately 5×
108/ml, which was about 10 times higher than when grown in an in vitro nutrient medium in a carbon dioxide gas incubator. These cells were suspended in the same manner as in Example A-2, and to this suspension was added Niekhanosul's disease virus with a hemagglutination value of about 500 per ml, which had been inactivated in advance with ultraviolet light, and further phytohemagglutinin was added at about 50μ per d. ? Add new lymphokine 1 by keeping at 37℃ for 1 day.
was induced to be generated.

Thereafter, it was purified, concentrated and dried in the same manner as in Example A-2 to obtain a new Nohokaino 1 powder.

Activity yield was approximately 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.
I kept it for a week. After the eggs were broken, proliferating cells were collected. This cell was 5X1o6/+ in the same manner as in Example A-1.
Suspended at t/l. Sendai virus with a hemagglutination titer of about 500 per ml was added to this suspension, kept at 37°C for 1 day to induce the production of It 177hocain 1, and then purified in the same manner as in Example A-2, concentrated and dried. teilJ・
Powder of Nohocaine ■ was obtained.

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

Example B-1 Injection Solution Fresh/Hocain IF 500. prepared in Example A-2.
000 units is dissolved in 200M physiological saline and aseptically filtered using a membrane filter. The filtrate was filled into two sterilized glass containers and lyophilized, and the containers were sealed tightly to obtain a lyophilized powder preparation.

This product is suitable for the treatment of breast cancer, lung cancer, liver cancer, leukemia, etc.

Example B-2 Injection In the production of the injection of Example B-1, 500.000 units of new nohokine II and lymphoblastoid cell-derived α-inter 7 elono aoo, ooo, ooo
A lyophilized powder preparation was obtained in the same manner as in Example B-1, except that the unit was dissolved in 200 ml of physiological saline.

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

Example B-8 Ointment The new lymphokine ■ prepared in Example A-8 was dissolved in a small amount of liquid paraffin according to a conventional method, and petrolatum was added to it.
It was made into an ointment with a concentration of 0.000 units/l.

This product is suitable for treating skin cancer, breast cancer, lymphoma, etc.

Example B - 4 points Eye preparation Distilled water 5 ooy Tome phenylethyl alcohol 5 m
1 and the new lymphokine ■ prepared in Example A-4,
NaCl was added to make the tonicity to 1.000+nl with one drop of distilled water to give an eye drop.

This product is suitable for the treatment of retinoblastoma, etc.

Example B-5 Enteric Coated Tablet When the new lymphokine ■ prepared in Example A-7 was tableted using a mixture of starch and maltose according to a conventional method,
New Lymphokine ■ 1 tablet (1oo1n9) of υ20
Tablets were prepared by containing 0.000 units of methyl cellulose phthalate, and the tablets were coated with methylcellulose phthalate to obtain enteric-coated tablets.

This product is suitable for the treatment of colorectal cancer, colon cancer, liver cancer, etc.

Claims (1)

[Claims] (1) Physicochemical properties: [1] Molecular weight 20,000±2,000 [2] Isoelectric point pI = 6.2±0.3 [3] Mobility Disc-PAGE , Rf=0.29±0.02[4
] Ultraviolet absorption spectrum with maximum absorption around 280 nm [5] Soluble in solvents Soluble in water, physiological saline or phosphate buffer Slightly soluble to insoluble in ethyl ether, ethyl acetate or chloroform [6] Color reaction Lowry method or shows a positive reaction for protein by the microburet method and a positive reaction for carbohydrates by the phenol-sulfuric acid method or the anthrone-sulfuric acid method [7] Effect: shows cytotoxic activity against L_9_2_9 cells;
Substantially exhibits no cytostatic activity or interferon activity against cells [8] Activity stability in aqueous solution: Stable up to 60°C when kept at pH 7.2 for 30 minutes; pH 4.0 when kept at 4°C for 16 hours. Stable in the range of 0 to 11.0 [9] A new lymphokine II that is stable for more than 1 month when stored frozen at -10°C. (2) The physical and chemical properties produced by applying an inducing agent to human-derived cells are as follows: [1] Molecular weight: 20,000 ± 2,000 [2] Isoelectric point pI = 6.2 ± 0.3 [3] Mobility Disc-PAGE, R_f=0.29±0.02 [
4] Ultraviolet absorption spectrum with maximum absorption around 280 nm [5] Solubility in solvents Soluble in water, physiological saline or phosphate buffers Slightly soluble or insoluble in ethyl ether, ethyl acetate or chloroform [6] Color reaction roller It shows a positive protein reaction by the phenol-sulfuric acid method or the microburet method, and a carbohydrate-positive reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method.
Substantially exhibits no cytostatic activity or interferon activity against cells [8] Activity stability in aqueous solution: Stable up to 60°C when kept at pH 7.2 for 30 minutes; pH 4.0 when kept at 4°C for 16 hours. Stable in the range of 0 to 11.0 [9] A method for producing new lymphokine II, which comprises collecting new lymphokine II that is stable for one month or more when stored frozen at -10°C. (3) Physical and chemical properties: [1] Molecular weight 20,000±2,000 [2] Isoelectric point pI = 6.2 ± 0.3 [3] Mobility Disc-PAGE, R_f = 0.29 ±0.02[
4] Ultraviolet absorption spectrum with maximum absorption around 280 nm [5] Solubility in solvents Soluble in water, physiological saline or phosphate buffers Slightly soluble or insoluble in ethyl ether, ethyl acetate or chloroform [6] Color reaction roller Shows a positive protein reaction by the phenol-sulfuric acid method or the microburet method, and shows a carbohydrate-positive reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method [7] Effect: Shows cytotoxic activity against L_9_2_8 cells;
Substantially exhibits no cytostatic activity or interferon activity against cells [8] Activity stability in aqueous solution: Stable up to 60°C when kept at pH 7.2 for 30 minutes; pH 4.0 when kept at 4°C for 16 hours. Stable in the range of 0 to 11.0 [9] New lymphokine II, which is stable for more than 1 month when stored frozen at -10°C, is purified using a monoclonal antibody specific for it and collected. A method for producing the new lymphokine II as defined in claim 2. (4) Physical and chemical properties: [1] Molecular weight 20,000±2,000 [2] Isoelectric point pI = 6.2 ± 0.3 [3] Mobility Disc-PAGE, R_f = 0.29 ±0.02[
4] Ultraviolet absorption spectrum with maximum absorption around 280 nm [5] Solubility in solvents Soluble in water, physiological saline or phosphate buffers Slightly soluble or insoluble in ethyl ether, ethyl acetate or chloroform [6] Color reaction roller It shows a positive protein reaction by the phenol-sulfuric acid method or the microburet method, and a carbohydrate-positive reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method.
Substantially exhibits no cytostatic activity or interferon activity against cells [8] Activity stability in aqueous solution: Stable up to 60°C when kept at pH 7.2 for 30 minutes; pH 4.0 when kept at 4°C for 16 hours. Stable in the range of 0 to 11.0 [9] A composition comprising a new lymphokine II that is stable for one month or more when stored frozen at -10°C. 5) The composition according to claim 4, characterized in that the composition contains other lymphokines together with the new lymphokine II. (6) The composition according to claim 5, wherein the other lymphokine is interferon. (7) The composition according to claims 4, 5, and 6, wherein the composition is an antitumor agent.