JPS60126228A - Novel lymphokine i, production and use thereof - Google Patents

Abstract

NEW MATERIAL:The novel lymphokine I having the following characteristics. Molecular weight, 20,000+ or -2,000; isoelectric point, pI=5.6+ or -0.2; mobility, Rf= 0.29+ or -0.02 by Disc-PAGE; solubility, soluble in water, saline water, etc., and hardly soluble or insoluble in ethyl ether, etc.; color reactions, positive to protein by Lowry method, etc., positive to saccharide by phenolsulfuric acid method; biological activity, exhibits proliferation suppressing effect to KB cell and cytotoxicity to L929 cell, and is essentially free from interferon activity; stability of the activity in aqueous solution, stable at 60 deg.C for 30min at 7.2pH, stable at 4 deg.C for 16hr at 4.0-11.0pH, unstable by dispase treatment, stable for >=1 month by freeze-storage at -10 deg.C. USE:An antitumor agent. PREPARATION:The novel lymphokine I can be prepred by treating a human- originated cell (e.g. leucocyte) capable of producing the novel lymphokine I with an inducing agent.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new lymphokine I having cytotoxic activity against tumor cells, and its production method and uses.

Lymphokines that have cytotoxic activity against tumor cells include lymphotoxin and Tumoa necrosis factor.

Lymphotoxins are co-authored by Takashi Aoki, “Lymphoid Parakeet New Immunology H'fJ 6 (1979), Igaku Shoin, former O.
om B, R, & Glade P, R, co-edited r I
n Vitr.

methods in cell-mediated
immunity JAcademicpress
(1,971) and Ce1lular Immun
ology Vol. 138. pp. 388-402 (1978
The Tumoa necrosis factor is described in Carswell F, A, et al., Pr.
Natl, Acad, Sci,, U, S, A,
, VoL 72, No., pp. 93666-3670 (1
975) and E, Pickta'l'umorNe
crosis Factor in Lymphokia
nes VoL L 23 to 272 pages, Academy
c Press (1981), etc.

Furthermore, recently, Haruo Onishi et al. 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 1 that has physicochemical properties that are completely different from those of previously known lymphokines, established a method for its production, confirmed cytotoxic activity against various malignant tumor cells, and discovered its uses. and completed the present invention.

That is, the present invention has physical and chemical properties such as (i) molecular weight of 20, QQO±2. QOO ■Isoelectric point pI=5.6±0,2 ■Mobility Disc -PAGE, Rf=0.29±0.0
2 ■ Ultraviolet absorption spectrum with maximum absorption around 280 nm ■ Solubility in solvents Soluble in water, physiological saline or phosphate buffer. Poorly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform ■ Effects of showing a positive protein reaction in the color reaction Lowry method or microburet method and positive carbohydrate reaction in the phenol sulfuric acid method or anthrone sulfuric acid method (I) It exhibits cytotoxic activity against 13 cells and L9°9 cells, and virtually no interferon activity ■ Activity stability in aqueous solution: Stable up to 0°C when maintained at pl-I7.2 for 30 minutes; Conditions for holding at 4°C for 16 hours: pH 4,,
Stable in the range of 0 to 10.0, dispase (DISP)
The present invention relates to new lymphokine 1 (this substance is referred to as new lymphokine 1 throughout this specification), which is unstable by ASE) treatment and is stable for one month or more when stored frozen at -10°C, and its production method and uses.

The method for producing new lymphokine 1 includes human-derived cells having the ability to produce new lymphokine 1, such as leukocytes, lymphocytes,
It may be produced by acting on 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 inoculated into a diffusion chamber, and the supply of body fluids of the warm-blooded animal is increased. It is preferable to multiply while receiving.

In other words, unlike in vitro cell proliferation, there is no need for expensive nutrient media containing serum, etc., or it is not a waste of money, and maintenance and management during cell proliferation is extremely easy. Moreover, it has the characteristic of having high activity as a new protein that is induced and produced from the obtained cells.

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 that can receive the animal's body fluids is implanted in the animal's body and the animal is reared normally, the body fluids containing nutrient ffj supplied from the warm-blooded animal's body can be used to diffuse its cells. can easily proliferate.

Compared to when cells are grown in vitro, W has shown that the growth of these cells is stable, the growth rate is high, and the amount of cells obtained is large; Another major feature is that the yield of new lymphokine I is significantly increased.

The cultivated human-derived cells used in the present invention need only 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 lymphokines. , for example, "Protein Nucleic Acid Enzyme Vol. 1.20, No.
Various human-derived cell lines described on pages 64616-643 (1975) can be used. Toshiwake,'r Journal of ClinicalM
icrC11nica1 Vol, I J 116~
Namalv described on page 117 (1975)
a cell, ■, Miyoshi r Nature V
ol, 267 J pp. 843-844 (1977)
BALL-1 cells, TALL-1 cells, NALL-1 cells, r Journal of Im
Munology Vol.

M-7002 cells and B-7101 cells described in 113 J pp. 1334-1345 (1974), "Tissue Culture" Vol. 6, No. 13, pp. 527-546 (1980)
JBL cells, EBV-8a cells, EB described in
V-wa cells, EBV-HOa cells, MOLT-3 cells, BALM2 cells, CCRF-8B cells (ATC
C'CCL 120) CCRF-CEM cells, DND-
Established lymphoblastoid cell lines such as 41 cells, and
Normal mononuclear cells, granular white blood cells, etc. are infected with various viruses,
Cells that have been treated with drugs, radiation, etc. and cultured are suitable.

In addition, we have developed a method of cell fusion using, for example, polyethylene gelcoal or Sendai virus, to transform the genes capable of producing new phosphokines in these human-derived cells.
N A IJ gauze, restriction enzyme (nuclease), DN
Cells may be treated with genetic recombination means using enzymes such as A polymerase to increase their proliferation rate, or cells may be used by increasing their ability to produce new lymphokine 1, as described herein. It is not limited only to established cell lines. These cells can be freely used alone or in combination of two or more types in the process of inducing and producing new lymphokine I, 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 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, pigeons, horses, cows, guinea pigs, rats, nude rats, hamsters,
Mammals such as normal mice and nude mice can be used.

Transplanting human-derived cells into these animals may cause unfavorable immune reactions, so in order to minimize such reactions, the animals used are kept as young as possible, i.e. eggs, embryos, fetuses, or newborns. Preferably those from childhood.

Alternatively, 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 to weaken the immune response before transplantation. 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.
This is particularly advantageous because cultured human-derived cells can be transplanted and rapidly proliferated without requiring these pretreatments.

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. In addition to stabilizing the proliferation of the derived cells, it is also possible to increase the amount of new lymphokine I induced therefrom.

In this case, transplantation may be carried out not only between the same species or between cohabitants, but also between cages or between the same 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,
Porous filtration membranes capable of blocking the passage of animal cells, such as membrane filters with a pore diameter of about 10-7 to 10-5m, known in various shapes and equipped with ultrafiltration membranes or follower fibers, with large diffusion chamber inside the animal body,
For example, it is implanted in the peritoneal cavity, and any of the human-derived cells cultured in the culture strain described above can be grown in the chamber while being supplied with body fluids containing nutrients from the animal body.

In addition, if necessary, a chamber that connects and perfuses the solution containing nutrients in this chamber/bar with body fluids within the animal body is attached to the surface of the animal body, and human-derived cells in the chamber are allowed to proliferate. The condition can be seen through a window provided on the surface of the chamber, and only a portion of this chamber can be attached and detached to allow cells to proliferate to the fullest lifespan without slaughtering the animal, thereby increasing cell production per animal. The amount can also be increased further.

Methods using these diffusion chambers do not only allow human-derived cells to be easily collected because human-derived cells do not come into direct contact with animal cells, but there is also less risk of causing an undesirable immune reaction, so there is no need to worry about immune reactions. There is no need for pre-treatment to suppress the disease, 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.

It has also been found that the number of human-derived cells obtained in this manner reaches about 107 to 1012 or more per animal.

In other words, the number of human-derived cells grown in vivo in an animal reaches about 102 to 107 times the number of cells transplanted into the animal's side, or more. This is about 101 to 106 times higher than that when multiplied, or even more, which is extremely convenient for the production of new lymphokines.

Any method can be used to produce the new 'J lymphokine It-induced 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, a neolymphokine 1 inducer is directly applied to human-derived cells grown in suspension in ascites in the peritoneal cavity or tumor cells generated subcutaneously to induce the production of neolymphokine ■, and then the serum, New lymphokine I may be purified and collected from ascites or tumor.

In addition, human-derived proliferating cells can be removed from the body of a non-human animal and treated with a new lymphokine-inducing agent in vitro to induce the production of new lymphocytes. For example, collecting human-derived cells proliferated in ascites, or excising a +lI+i tumor containing human-derived cells generated subcutaneously,
The resulting cells are suspended in a nutrient medium maintained at approximately 20 to 40°C to a cell concentration of approximately 105 to 1.08/ld, and a new lymphokine I inducer is applied to induce the new lymphokine. ): It can be produced by induction 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 inducing agent to induce the new lymphokine, ■. It can also be generated.

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 lymphokine 1 by applying a sunhokine I inducer.

In addition, for example, first, neolymphokine 1 is induced to be produced in the animal body in grown 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 body. A method of inducing the production of new lymphokine ■, or a method of using cells once used for the induction production of new lymphokine I for the induction production of new lymphokine I two or more times, or embedding or connecting them in an animal body. It is also possible to further increase the amount of new phosphokine used on the animal's body by increasing the number of cells obtained by exchanging the chamber.

The new lymphokine 1 inducer of the present invention includes viruses, nucleic acids, and
nucleotides, phytohemagglutinin, and concanavalin A, which are known as γ-interferon inducers.
1. Borkwiet mitogen, ribopolysaccharide, endotoxin, polysaccharide, bacteria, etc. are used as appropriate. Antigens are also inducers of neolymphokine 1 for sensitized cells.

Furthermore, when producing new lymphokine I from human-derived cells, the production amount of new lymphokine I can be increased by using an α-interferon inducer and a γ-interferon inducer together as a new lymphokine inducing agent. is also free.

Furthermore, it has been found that not only new lymphokine I is 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 1 induced and produced in this way can be easily purified and separated and collected by performing known purification and separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and freeze-drying. can. 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-performance liquid chromatography, column chromatography, and affinity chromatography, the highest purity new lymphokine 1 can be collected. It is also possible.

Furthermore, the new lymphokine r thus obtained can be used as an antigen to immunize a warm-blooded animal other than humans, collect antibody-producing cells from the animal, and fuse these cells with myeloma cells. Select fused cells that have the ability to produce anti-lymphokine antibodies from the fused cells, grow the selected cells, and purify the produced monoclonal antibody using, for example, an immobilized monoclonal antibody obtained by reacting with bromcyan-activated Sepharose. However, it can also be advantageously used to collect highly purified new lymphokine (2) at a high yield.

The new sumpocaine 1 purified and manufactured in this way is
physical and chemical properties.

■Molecular weight 20.000±2,000 ■Isoelectric point p I = 5.6±0.2 ■Mobility Disc-PAGE, Rf = 0.29 + 0.02
■ Maximum absorption in the ultraviolet absorption spectrum around 280 nm ■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer.

Slightly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform ■ Effect of showing a positive protein reaction in the color reaction Lowry method or microburet method, and a positive carbohydrate reaction in the phenol sulfuric acid method or anthrone sulfuric acid method K B Shows cytotoxic activity against cells and L9□9 cells, and virtually no interferon activity ■ Activity stability in aqueous solution: Stable up to 0°C when kept at pH 7.2 for 30 minutes, stable at 4°C Stable in the pH range of 4.0 to 10.0 under conditions of 16 hour retention
) Unstable due to treatment ■ It was found to be stable for more than one month when stored frozen at -10°C.

It has also been found that new lymphokine I has the ability to damage and kill not only mouse myeloma cells but also various human tumor cells, but does not substantially damage normal human cells.

Therefore, new lymphokine 1 can be used as a composition containing it to treat new lymphokine I-sensitive diseases, such as preventive and therapeutic agents for malignant tumors, including various human malignancies that have been considered extremely difficult to treat. It can be advantageously used as a tumor therapeutic agent.

The activity of new lymphokine I is influenced by KB cells and KB cells as target cells.
or L9□, measured using cells. That is, when using KB cells, Cancer Chemother
apy1'(eports parts 3. Vol.
,3,No,2. The growth inhibitory activity of KB cells was measured according to the description in September 1972, and L9
□When using 9 cells, see E. Pick, Tum.
or Necrosis Factor in” Ly
mphokines' Vol, B%pP, 245
~249, Academic press (1981
L in the presence of actinomycin D as described in
Cytotoxic activity on 9□9 cells was measured. In the present specification, an activity measurement method using %L, 29#lIl cells was adopted, unless otherwise specified.

The activity of interferon, which is highly species-specific to humans, is described in “Protein Nucleic Acid Enzyme J Vol. 20. No. 6.616
It was measured by the known plaque half-life method using human amniotic membrane-derived FL cells as reported in 1975, p.

The hemagglutination titer was determined by J. F. 5alk's Journal.
al of Immunology JVoL 49.8
The measurement was carried out according to the method of P. 7 (1944).

Next, the present invention will be explained through experiments.

Experiment A-1 Preparation of Partially Purified New Popokine I Neonatal hamsters were injected with antiserum prepared from rabbits by a known method to weaken the immune response of the hamsters, and then BALL-1 was injected subcutaneously into the hamsters. The cells were transplanted and then reared for 3 weeks in the usual manner. 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.
and suspended in the same medium at a density of approximately 2 x 106/m6. Approximately 400 cells per #+ε for this cell suspension
Add Sendai virus with hemagglutination titer L, 2 at 37°C.
The cells were kept for 4 hours to induce generation of new lymphokine I.

This was heated to about 4°C and about 1. ．． The supernatant obtained was centrifuged at 000 g to remove the precipitate, and the resulting supernatant was dialyzed against physiological saline containing pH 7.2.0 OLM phosphate buffer for 20 hours, followed by microfiltration. The filtrate was passed through an antibody column immobilized with anti-interferon antibodies, the non-adsorbed fraction was collected, and this was then subjected to chromatofocusing.The live blood fraction was collected, concentrated, and lyophilized. A powder containing new Sunhokine I activity was obtained.

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

Furthermore, the yield of new lymphokine I was approximately 30 million years per hamster.

Experiment A-2 Preparation of Anti-Folding Lymphokine I Antibody The new lymphokine I obtained by the method of Experiment A-1 was dissolved in physiological saline to a protein concentration of approximately 0.05 w/v. Mix equal amounts of adjuvant emulsion,
This mixture @02m1 was injected subcutaneously into the mice, and 7 days later, the mice were immunized by the same injection again. Anti-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 P3-X63-Ag8 (F
IOW I (manufactured by laboratories) at 50 w/v prepared in serum-free Pagle's minimal basal medium.
% polyethylene glycol-1000 solution (p) (7,
2. Temperature: 37°C), each was suspended at a concentration of 104/- for 5 minutes, diluted 20 times with the basic medium, and then Somatic
Ce1l Genetics, VOI.

2. Collect fused cells that can proliferate in hypoxanthine-aminopterin-thymidine culture solution according to the method reported on pages 175-176 (1976), and have the ability to produce anti-lymphokine 1 antibodies from these fused cells. Fused cells were selected. Approximately 106 of the obtained fused cells were transplanted into the peritoneal cavity of each mouse and raised for 2 weeks.The mice were then sacrificed and body fluids such as ascites and blood were collected, centrifuged, and the supernatant was precipitated with ammonium sulfate. The precipitated fraction with a saturation degree of 30-50% was collected and then dialyzed, and this liquid was further reacted with the new lymphokine It bromocyanically activated sepharose obtained by the method of Experiment A-1 at room temperature. Affinity chromatography was performed using immobilized new lymphokine I gel to obtain anti-refractory lymphokine I antibody fractions, which were dialyzed, concentrated, and lyophilized to collect new lymphokine I monoclonal antibody powder.

Motoichi demonstrated immunologically specific neutralizing activity against the cytotoxic activity of the new lymphokine ■.

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. The active fraction of the new lymphokine was collected by affinity chromatography using the gel.
Dialyzed, concentrated and lyophilized.

Motoichi was a highly purified new lymphokine, and its specific activity was approximately 109 units/Misaki protein. Motoichi also showed almost the same specific activity using an activity measurement method using KB cells.

Using Motoichi, we investigated the physical and chemical properties.

■For molecular weight, Weber and M, Osbom%J, Bi
ol, Chem, .

Vol. 244, p. 4406 (1969), 5DS-polyacrylamide gel electrophoresis was performed. That is, about 10 μg of a sample was loaded onto an ion acrylamide gel column in the presence of 0.196 SDS, and after 4 hours of electrophoresis at p 8 mA per column, it was extracted, and the molecular weight was determined from the activity measurement. 000
Met.

■Isoelectric point Gel for isoelectric focusing, manufactured by LKB, Sweden, product name: AMPHOLINE PAGPLATB (pH 3,
As a result of electrophoresis at 25W for 2 hours using 5-9.5),
Isoelectric point 1) I was 5.6±0.2.

■ Electrical mobility B, J, Davis, Ann-N, Y, Acad
, Set, , Vol, 121.404 pages (196
Approximately 10 μg of the sample was loaded onto a 7.5% acrylamide gel column, and after electrophoresis at pH 8.3 and 3 mA per column for 2 hours, it was extracted and the electromobility was determined from the activity measurement. However, Jo was 29±0.02.

■ Ultraviolet absorption spectrum Spectrophotometer manufactured by Shimadzu Corporation, product name UV-25
As a result of examining the absorption spectrum in the ultraviolet region for 0t-
Maximum absorption was observed near 280iBn.

■ Solubility in solvents Soluble in water, saline or phosphate buffer.

It was sparingly soluble to insoluble in ethyl ether, ethyl acetate, or chloroporum.

(2) Color reaction Lowry method or microburet method showed a positive protein reaction [7, phenol sulfuric acid method or anthrone sulfuric acid method showed a positive carbohydrate reaction.

■Effect: Cytotoxic activity was shown against KB cells and 29 cells. Virtually no interferon activity was shown.

■ Activity stability in aqueous solution l) Thermal stability A sample of approximately t x io5 units/- was subjected to various temperatures at pH 7.
, 2. After holding for 30 minutes, remove the remaining activity.
As a result of measuring cell damaging activity, it was stable up to 60°C.

+t) I) Add 0.1- of a sample with H stability of approximately I
r, pH7~B ・-Phosphat buffe
r, pH 8-11-Glycine-NaOHbu
29 cells. As a result of measuring the harmful activity against pH 4,
It was stable in the range of 0 to 10.0.

111) Stability towards DISPASE approximately I x 105 units/7! Dispase (grotease derived from Bacillus bacteria manufactured by Godo Shusei Co., Ltd.) was added to the sample at a concentration of io units/me, and the pH was adjusted to 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 new lymphokine I in this solution, it was found that new lymphokine (2) was stable upon treatment with dispase, and the activity of new lymphokine I was lost as the reaction progressed.

+V) Stability due to storage - After freezing at 10°C and storing for one month, the product was thawed and the activity was measured. As a result, no decrease in activity was observed.

From the above results, the new lymphokine ■ has clearly different physicochemical properties from the previously known lymphokines such as lymphotoxin and TNF1 interferon.

Experiment B-1 Growth inhibitory effect on malignant tumor cells Using the new lymphokine I obtained by the method of Experiment A-1 or Experiment A-3, the growth inhibitory effect on various human-derived cells was investigated.

After culturing io' cells of each type of human-derived cells for 9.1 days in a known nutrient medium 1- supplemented with fetal bovine serum, the new cells prepared by the method of Experiment A-1 or Experiment A-3 were added to the culture medium. Physiological saline 0.1- containing 50 units or 500 units of lymphokine l was added and cultured at 37°C for 2 days. After completion of culture, Applied Microbiology Vo
L22, No. 014, pp. 671-677 (1971), live cells were stained with the stain Neutral Red, the stain was subsequently eluted with acid ethanol, and the eluate was The amount of viable cells was measured from the absorbance at nm.

For the control experiment, 0.1 rnt of physiological saline not containing the new lymphokine (■) was used.

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

Cell proliferation inhibition rate (chi) = The results are shown in Table 1.

Table 1 As is clear from the results in Table 1, new lymphokine I was found to have almost no effect on normal cells, but to significantly suppress the proliferation of various malignant tumor cells. did. 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, the new lymphokine ■ obtained by the method of Experiment A-3 was dissolved in physiological saline. , once daily, 100 or 1ρOO units/Kg 1
Intravenous injections were given for 5 days. Thereafter, mice were sacrificed and tumor weights were measured.

The results are shown in Table 2.

* With a risk rate of 5% or less, there is no statistically significant difference compared to the control value.

Experiment B-3 A piece of human breast cancer tissue was subcutaneously transplanted into the back of a BAL B/C-derived nude mouse, and from the time when the tumor volume reached approximately 200 -, experiment A-1 or experiment A-3 was carried out. The Shinsunhokai/■ obtained by the method was dissolved in physiological saline and intravenously injected once daily at 100 or 1,000 units/Ky 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 3 * There is a statistically significant difference compared to the control value when the risk rate is 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 20-day-old mice.
The toxicity of the new lymphokine I 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 new lymphokine I of the present invention can be used not only in vitro but also in vitro.
It is also effective in inhibiting the growth of malignant tumors in vivo, and its stability is extremely high in terms of the mice in which it is used.

The daily dose for adults of the new lymphokine I of the present invention is 5 to 5.
500,000,000 units, preferably for topical applications such as local injections and eye drops, 5-! 10,000
ρ units, 10 to so, ooo, ooo units for transdermal or transmucosal applications such as ointments or suppositories, 50 to 100,000,000 units for systemic injections such as intravenous and intramuscular injections, and 50 to 100,000,000 units for oral administration. In this case, the dosage is 500 to 500,000,000 units, but it can be increased or decreased as appropriate depending on the 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. The amount used is New Lymphokine 1
Considering the toxicity, effective amount, and safety of the new lymphokine, it is desirable to contain 5 or more units of the new lymphokine per gram of the pharmaceutical preparation.

The form of a new preventive or therapeutic agent for a disease susceptible to lymphokine I containing polymphokine I 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. It can also be used as a nasal or eye drop, or as an ointment.

In addition, when treating a malignant tumor using new lymphokine 1, for example, a part of a patient's tumor is taken and treated with new lymphokine I to increase the immunogenicity of the tumor. By putting it back into the patient's body, it is also possible to treat this malignant tumor more effectively.

Hereinafter, Example A will describe a production example of the new popokine I of the present invention, and Example B will describe a production example of various drugs that are compositions of the new popokine 1 of the present invention.

Example A-1 Eagle's minimal basic medium (1) containing human-derived lymphoblastoid BALL-1 cells supplemented with 20% fetal bovine serum
H7,4) and in vitro (in vitro) at 37°C according to the usual method.
The cells were cultured in suspension (vitro). The obtained cells were washed with serum-free Eagle's minimal basic medium (pH 7.4) and suspended in the same medium to a concentration of approximately I x 10,'y. Approximately 1.0 ml of Sendai virus was added to this suspension.
000 blush sphere agglutination value was added and kept at 38°C for 1 day to induce the production of new lymphokine (■). This at 4℃, about i, oo
The supernatant obtained was centrifuged at pH 7,2,0,0.
The filtrate obtained by dialysis with physiological saline containing 1M IJ phosphate buffer for 15 hours and further microfiltration was applied to an anti-interferon antibody column in the same manner as in Experiment A-1, and the non-adsorbed fraction was The monoclonal antibody gel column prepared by the method of Experiment A-3 was subjected to affinity chromatography and concentrated to obtain a concentrated solution of new lymphokine I having a specific activity of about 109 units/■ protein.

The activity yield was approximately 2 million units per suspension 1 during induction production.

Example A-2 After injecting neonatal hamsters with antiserum prepared from rabbits by a known method to weaken the immune response of the hamsters,
Human-derived lymphoblastoid BALL-1 cells, which had been cultured, were subcutaneously transplanted into the mice, and then raised for 3 weeks in a conventional manner. Approximately 151 tumors generated under the skin were removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were washed with serum-free RPMI 1.640 medium (pH 7,2) and suspended in the same medium at approximately 5 x 106/me.
Ta. Approximately 1 per me of Sendai virus was added to this suspension.
000 hemagglutination titer and E, coti Yamaki endotoxin about 10μ per me? was added and kept at 37°C for 1 day to induce the production of new lymphokine (■). This is about 4℃
The precipitate was removed, the resulting supernatant was dialyzed for 21 hours in physiological saline containing pH 7, 2,0,01M IJ phosphate buffer, and further microfiltered. The obtained filtrate was purified using an antibody column in the same manner as in Example A-1, and the resulting solution was concentrated and lyophilized to obtain a new protein I having a specific activity of about 109 units/η protein. powder 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 for 5 weeks in a conventional manner. Into this abdominal cavity, about 3,000
Newcastle disease virus with a hemagglutination titer of 0 was previously inactivated by ultraviolet light 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 new lymphokine I.

The activity yield was approximately 4 million units per nude mouse.

Example A-4 After pre-irradiating a normal mouse with approximately 400 rem of X-rays to weaken the immune capacity of the mouse, the MOI of human-derived lymphoblastoid cells was cultured subcutaneously in the mouse.
IO-1 cells were transplanted and then reared for 3 weeks in the usual manner. After removing a tumor of about 1 of which had occurred under the skin, 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, Sendai virus with a hemagglutination value of about 500 per me and concanavalin A with a hemagglutination value of about 500 were added to this suspension, and the mixture was kept at 37°C for 1 day. New lymphokine 1 was induced to be produced. Thereafter, the product was purified, concentrated, and dried in the same manner as in Example A-2 to obtain a powder of Shinsunhokai/1.The activity yield was 24 million units per mouse.

Example A-5 Human-derived lymphoblastoid cells Namalvai cultured in newborn hamsters in the same manner as in Example A-2
The cells were transplanted and then reared in the usual manner for 4 weeks. A subcutaneous tumor of about 20 f was loosened in the same manner as in Example A-2 to obtain a cell suspension of about 3 x 10 6 /me. Sendai virus was added to this suspension to give a hemagglutination value of approximately 1,000 per portion, and the suspension was kept at 36°C for 2 days to induce the production of new lymphokine I. The suspension was then purified and concentrated in the same manner as in Example A-1 to produce new lymphokine. A concentrated solution of I was obtained.

The activity yield was approximately 26 million units per hamster.

Example A-6 Human-derived lymphoblastoid cells N cultured in a plastic cylindrical diffusion chamber with an internal capacity of about 10 me provided with a membrane filter with a pore size of 0.5 microns.
ALL-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/mg, which is about 102 times higher than when grown in an in vitro nutrient medium in a carbon dioxide gas incubator. The cells were suspended in the same manner as in Example A-2, and to this suspension - Newcastle disease virus with a hemagglutination value of approximately 500 was added, having been inactivated in advance with ultraviolet rays, and phytohemagglutinin was further added.
This comb is about 50μ? The cells were kept at 37° C. for 1 day to induce the production of new lymphokine I. Thereafter, it was purified in the same manner as in Example 2, concentrated and dried to obtain a whole powder of new lymphokine (2).

Activity yield was approximately 10 million units per rat.

Example A-7 Human-derived cell line CCRF-CEM cells were transplanted into fertilized chicken eggs that were kept at 37°C for 5 days, and then kept at 37°C for 1 week. After the eggs were broken, proliferating cells were collected. These cells were grown at 5 x 106 cells in the same manner as in Example A-1.
- 1 was suspended in. Sendai virus with a hemagglutination titer of about 500 per me was added to this suspension and kept at 37°C for 1 day to induce the production of new lymphokine ■, and then Example A
-2 was purified and concentrated to obtain a powder of new lymphokine 1.

The activity yield was approximately 800,000 units per 10 fertilized eggs.

Example B-1 Injection New Sunhokai prepared in Example A-2/1500.00
0 unit is dissolved in 200 me physiological saline and aseptically filtered using a membrane filter. Transfer the filtrate to a sterilized glass container at 2rn1. Each sample is filled, lyophilized, and sealed to form a lyophilized powder preparation.

This product is suitable for treating breast cancer, lung cancer, liver cancer, leukemia, etc.

Example B-2 Ointment New Lymphokine 1 prepared in Example A-3 was dissolved in a small amount of liquid paraffin according to a conventional method, and vaseline was added to it.
It was made into an ointment of 0,000 units/2.

The city is suitable for treating skin cancer, breast cancer, lymphoma, etc.

Example B-3 Eye Drops Steamed water 5 ml β-phenylethyl alcohol 5 ml
1 and the new lymphokine 1 prepared in Example A-4.
000,000 units of sodium chloride was added to make the solution isotonic, and the volume was made up to 1,000 tnl with distilled water to prepare eye drops.

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

Example B-4 Enteric Coated Tablet When compressing the new Lymphokine I prepared in Example A-7 into tablets using a mixture of starch and maltose according to a conventional method,
Tablets were prepared by containing TNF in an amount of 200 ρOO per tablet (100 cm), and were coated with methylcellulose phthalate to form enteric-coated tablets.

This product is suitable for treating colorectal cancer, colon cancer, liver cancer, etc.

Patent applicant procedural amendment dated December 15, 1980 1. Indication of the case Patent Application No. 233570 of 1988 2. Name of the invention New Lymphokine ■ and its manufacturing method and use 3. Person making the amendment Relationship with the case Patent Applicant 4. The "Scope of Claims" section of the specification band to be amended and 5. Contents of Amendment (1) "Scope of Claims" will be amended as shown in the attached sheet.

(2) Specification No. 7, lines 12-13 of the book [Co-authored by Takashi Aoki and others, “Lymphokine” New Party Epidemiology Series 6 (1979)
Igaku Shoin,” has been corrected to “Lymphokine, co-authored by Takashi Aoki et al., New Party Epidemiology Series, pp. 6.87-105 (1979), Igaku Shoin.”

(3) Correct "1 mobility" stated in the 19th line of the 8th Sada and the 1st line of the 21st Sada to ``Mobility.''

(11) 1 soluble as written and written on page 9, lines 4-5, and page 22, line 5. " is corrected to "soluble."

(5) Page 9, lines 12-13 and page 22, line 13
~14 lines of 1-K B +? IB cyst and L92
" exhibits cytotoxic activity against 929 cells," is corrected to "1 (exhibits cytotoxic activity against B cells, and f) exhibits cytotoxic activity against 929 cells."

(6) Stable in the range of l' pl-14,Q to 100 as described in page 9, line 18 and page 22, line 19.
is corrected to "stable in the range of pi-14.0 to 11.0."

(7) The phrase "produced into a cultured stock" described in page 111, item 16 of the same document will be corrected to "made into a single cultured stock."

(8) "Anti-lymphokine antibody production capacity" stated in line 7 of page 21 will be corrected to "anti-lymphokine I antibody production capacity."

(9) [M, Osbomj described on page A, line 5]
Correct to M, 0sborn, j.

(1 [Exhibited cytotoxic activity against l'-KB cells and L929 cells described in lines 19 to 20 of the 29th edition of the same publication.)
"L92 has cytostatic activity against KB cells and
9 shows cytotoxic activity against cells. ” and cut it out.

α1) Page 30, line 6 and line 15 [L92
9 cells with harmful activity] is deleted.

04 [phosphat b described in line 11 of the same @page
buffer, J as “Phosphate buffer”
, correct to J.

("It was stable in the pH range of 4.0 to 10.0." stated in 131, 30R, line 16 will be corrected to "It was stable in the pH range of 4.0 to 110.")

α荀 "iv) Stability due to storage" stated in line 10 of page 31 of the same document will be corrected to "■ Stability due to frozen storage."

(151, page 31, line 11, “at -10°C”)
Correct the aqueous solution of PTL 7.2 to J at -10℃.

(16) 3rd] jff1m15 lines, '+llO
l"' TNF J Wol-7-E: Correct to 7 necrosis factor (TNF) J.

f17) [Liver
Correct chang J to l Chang Liver J.

(1) ``Experiment A-'' described on page 38, lines 14-15
[Prepared by the method described in Experiment A-3]
will be corrected.

OI ``Concentrate'' on page 43, lines 12-13 will be corrected to ``concentrate and dry''.

(JK is corrected using the method in Example A-2 for example A-2.

(21) "In Example A-3" written in page 44, line 6 of the same document is amended to "Actually not (by the method of Example A-3)".

(2) “In Example A-4” described in page 44, line 13 of the same
Correct it to "by the method of Example A-4".

I2 Co., page 14, lines 18-20
-7 = 1 tablet (1 tablet of New Lymphokine 1 prepared by the method of Example A-7)
(per 100mg).

2. Scope of claims (1) Physical and chemical properties: ■Molecular weight 20, OOO±2. OOO ■Isoelectric point pI=5.6±02 ■L Emperor” 1) With isc-PA01 bow, 1tf=0.29±0.0
2 ■ Maximum absorption in the ultraviolet absorption spectrum around 280 m ■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer, poorly soluble or insoluble in ethyl ether, ethyl acetate, and chloroform ■ Color reaction roll It shows a protein-positive reaction by the -method or microburette method, a carbohydrate-positive reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method, and it shows cytotoxic activity against L929 follicles, substantially inhibiting interferon activity. ■Activity stability in aqueous solution Stable up to 60℃ by holding at pH 7.2 for (9) minutes, pf-140 by holding at 4℃ for 16 hours
Stable in the range of liquid to plate
) Unstable due to processing ■ New/Hocaine I that is stable for more than 1 month when stored frozen at -10°C.

(2) The physical and chemical properties produced by causing human-derived S cells to emit induced molecules are: ■Molecular desire 20.000±2,000 ■Isoelectric point pi==5.6±0.2 ■ Mobility Disc-PAGE, f = 0.29 ± 0.0
20 Ultraviolet absorption (Subek)/Maximum absorption around 2800 m■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer Slightly soluble to insoluble in ethyl ether, ethyl acetate, or chloroporm■ Color reaction Lowry method Or, it shows a protein-positive reaction by the microburet method and a carbohydrate-positive reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method. ■Activity stability in aqueous solution Stable up to 66°C when held at pil, 2 for 16 hours; pf-14.0 to 14.0 when held at 4°C for 16 hours. Dispase (1) ISP stable at 1 level of 11,0
A method for producing new lymphokine I, which is characterized by collecting new lymphokine I which is unstable due to ASE) treatment and is stable for one month or more when stored frozen at -10°C.

(3) Physical properties (l-): ■Molecular weight 20.000±2,000 ■Isoelectric point pI=5.6±0.2 ■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 microview It shows a positive protein reaction by the Rett method and a positive carbohydrate reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method.It shows cytostatic activity against KB cells, cytotoxic activity against L929 cells, and interferon activity. Activity stability in aqueous solution Stable at ωt4 by holding at pH 7.2 for 130 minutes, stable at pH 4.0 to ) I S i'
A, S E) Unstable due to treatment - New lymphokine I, which is stable for more than 1 month when stored frozen at ℃, is purified using a monoclonal antibody specific for it and collected. A method for producing new lymphokine 1 according to claim 2.

(4) The physical and chemical properties are ■Molecular weight 20.000±2,000 ■Isoelectric point p■=5.5±0.2 ■''2] L-skin 1) isc-PAOK, 几f=0. 290.02■
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, and chloroform ■ Color reaction Lowry method or It shows a protein-positive reaction by the microburet method and a carbohydrate-positive reaction by the phenol-sulfuric acid method or anthrone-sulfuric acid method. It shows cytostatic activity against KB cells and cytotoxic activity against L929 regular cells. , shows virtually no interferon activity, ■ Activity stability in aqueous solution: Stable up to 60°C when maintained at pI-17.2 for addition, pH 4.0 to 11.0 when maintained at 4°C for 16 hours. Stable within the range of DISPAS
E) Unstable upon processing ■ A composition characterized in that it contains a new lymphokine I that is stable for more than one month when stored frozen at -1O<0>C.

(5) The composition according to claim 4, wherein the composition is an antitumor agent.

Claims (1)

[Claims] (11 Physical and chemical properties are: ■Molecular weight 20.000±2,000 ■Isoelectric point pJ=='56±0.2 ■Mobility 1) In isc-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 or insoluble in ethyl ether, ethyl acetate, or chloroform ■ Shows a positive protein reaction using the color reaction Lowry method or Miku Shiro Buret method, and shows a positive carbohydrate reaction using the phenol sulfuric acid method or anthrone sulfuric acid method KB Shows cytotoxic activity against cells and 29 cells, and virtually no interferon activity ■ Activity stability in aqueous solution Stable up to 60°C when kept at pH 7.2 for 30 minutes, 16 hours at 4°C Stable in the pH range of 4.0 to 10.0 depending on the conditions in which it is maintained, DISPAS
E) Unstable due to processing ■ New lymphokine ■ 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: ■Molecular weight: 20.000±2,000 ■Isoelectric point pI=5.6±0.2 ■Mobility: pisc-PAGE , J = 0.29±0.02
■ Maximum absorption in the ultraviolet absorption spectrum around 280 nm ■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer. Slightly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform ■ Shows a positive protein reaction using the color reaction Lowry method or microburet method, and shows a positive reaction for carbohydrates using the phenol-sulfuric acid method or anthrone sulfuric acid method K B Shows cytotoxic activity against cells and L9□9 cells, and virtually no interferon activity ■ Activity stability in aqueous solution: Stable up to 0°C when kept at pH 7.2 for 30 minutes, stable at 4°C Stable in the pH range of 4.0 to 10.0 under conditions of 16 hour retention, dispase (DI 5PA)
SE) A method for producing new lymphokine I, which is characterized by collecting new lymphokine I which is unstable due to treatment and is stable for one month or more when stored frozen at -10°C. (3) Physical and chemical properties: ■Molecular weight 20.000±2,000 ■Isoelectric point pI = 5.6±0.2 ■Mobility Disc-PAGE, Rf=0.29±0.02■
Maximum absorption in the ultraviolet absorption spectrum around 280 nm ■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer. Slightly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform ■ 0-effect KB cells that show a positive protein reaction by the color reaction Lowry method or microburet method, and a positive carbohydrate reaction by the phenol sulfuric acid method or anthrone sulfuric acid method and L9□, exhibiting cytotoxic activity against cells and virtually no interferon activity ■ Activity stability in aqueous solution: Stable up to 0°C when kept at pH 7.2 for 30 minutes; stable at 4°C up to 16°C 1) Stable in the range of H4.0 to 10.0, Dispase (DISPAS)
E) Cine stability due to treatment ■ A patent characterized by purifying and collecting new lymphokine 1, which is stable for more than one month when stored frozen at -10°C, using a monoclonal antibody that is specific for this lymphokine. A method for producing new lymphokine 1 according to claim 2. (4) The physical and chemical properties are: ■Molecular weight 20.000±2,000 ■Isoelectric point pI=5.6±0.2 ■Mobility Disc-PAGE, R4=0.29±0.02■
Maximum absorption in the ultraviolet absorption spectrum around 280 nm ■ Solubility in solvents Soluble in water, physiological saline, or phosphate buffer. Poorly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform ■ Effects of KB cells that show a positive protein reaction by the color reaction Lowry method or microburette method, and a positive carbohydrate reaction by the phenol sulfuric acid method or anthrone sulfuric acid method It exhibits cytotoxic activity against L9□9 cells and virtually no interferon activity ■ Activity stability in aqueous solution: Stable up to 0°C when kept at pH 7.2 for 30 minutes, stable at 4°C up to 16 Stable in the pH range of 4.0 to 10.0 depending on the conditions of time retention, dispase (DISPASE)
) A composition characterized by containing a new lymphokine (2) which is unstable upon treatment (1) and which is stable for one month or more when stored frozen at -10°C. (5) The composition according to claim 4, wherein the composition is an antitumor agent.