JPS61115028A - Agent for promoting antitumor effect - Google Patents

Abstract

PURPOSE:An agent for promoting antitumor effect, containing novel lymphokine II. CONSTITUTION:The objective agent for promoting the antitumor effect can be prepared by using a novel 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 ethyl ether, 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) as an active component. 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 abn inducing agent. The lymphokine is essentially harmless to the normal human call.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an anti-tumor effect enhancer containing as an active ingredient Shinsunhokai/■ which exhibits cytotoxic activity against tumor cells; Regarding enhancers.

Generally, as chemotherapeutic agents for malignant tumors, alkylating agents, antimetabolites, antitumor antibiotics, plant alkaloids, and the like are used alone or in combination of two or more.

However, chemotherapeutic agents have drawbacks such as excessive side effects on patients, a narrow antitumor spectrum, and a tendency to induce drug-resistant tumors.

The present inventors have continued research into antitumor effect enhancers for chemotherapeutic agents, focusing on lymphokines, with the aim of improving these drawbacks of chemotherapeutic agents.

Conventionally, lymphokines having cytotoxic activity against tumor cells include lymphotoxin and tumor necrosis factor.

Photoxin is a ``lymphokine'' co-authored by Takashi Aoki et al.
New Party Epidemiology Series 6.87-105 (1979) Igaku Shoin, Bloom B.
R,) and Blade P,R
Co-edited with In vitro methods in cell-mediated immunity (In vitro methods in cell-mediated immunity)
thods 1ncell -mediated im
community ) J Academic Press (Acad
emic Press) (1971) and [Cellular Immunology
38, pp. 388-402 (19
1978), and the Tsumore Necrosis Factor was described by Carswell E.
E, A, ) etc., [Pro/-D/Guess Off the
Proceedings of the National Academy of Sciences
the National Academy ofSc.
72, No. 9, pp. 3666-3670 (1975) and Edited by E. Pick [Tsumoa Necrosis]
Factor in Lymphokines (Tumor Ne
crosis Factor in Lymphokin
es) J Vol. 2, pp. 235-272, Academic Press (1)
981), etc.

The inventors have been studying lymphokines for many years. As a result, we recognized the existence of a new lymphokine, which has physicochemical properties completely different from those of previously known lymphokines. The present invention was established by confirming the enhanced anti-tumor effect against cancer and establishing its use.

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 Disc-PAGE Rf = 0.29±0. 02■
Ultraviolet absorption spectrum Maximum absorption around 280 nm ■ Soluble in solvents Soluble in water, physiological saline or phosphate buffer Slightly soluble or insoluble in ethyl ether, ethyl acetate or chloroform ■ Color reaction Lowry method or microburet It shows a protein-positive reaction by the phenol-sulfuric acid method and a carbohydrate-positive reaction by the anthro/sulfuric acid method. It shows cytotoxic activity against L929 cells, and substantially inhibits cell proliferation and interferon activity against KB cells. ■Activity stability in aqueous solution Stable up to (a) ℃ by holding at pH 7.2 for an addition period; Stable in the pH range of 4.0 to 11.0 by holding at 4℃ for 16 hours■ - The present invention relates to an antitumor effect enhancer containing a new lymphokine (hereinafter referred to as new lymphokine) which is stable for one month or more when stored frozen at 10°C.

The method for producing new lymphokine H is, for example, new lymphokine I.
It may be produced by causing an inducer to act on human-derived cells capable of producing I, such as leukocytes, lymphocytes, and cultured cells.

Human-derived leukocytes and lymphocytes may be prepared by separating blood collected from humans.

The cultured human-derived cells can be used as cells grown in vitro according to a conventional method.

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.

That is, unlike the case of in vitro proliferation, not only is there no need for a trophic medium containing expensive serum etc., or can be greatly reduced, but maintenance and management during cell proliferation is also extremely easy. Furthermore, the new lymphokines produced by the obtained cells are characterized by high activity.

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 inside the animal's body and the animal is reared normally, its cells can be grown using the nutrient-containing body fluids supplied by the warm-blooded animal's body. They can easily multiply.

Furthermore, compared to the case of in vitro 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 new cells per cell is Another major feature is that the yield of 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 lymphocytes, such as ""Protein Nucleic Acid Enzyme" Volume Kan, No. 6, 616-
Various human-derived cell lines described on page 643 (1975) can be used. Among others, [Journal of Clinical Microbiology (Jo
Urnal of Clinical Microbio
(1975) J Vol. 1, pp. 116-117 (1975
Namalva cells, Imy 1, described in
7 (I, Miyoshi) [Nature
re ) J Vol. 267, pp. 843-844 (1977
BALL-1 cells, TALL-1, described in
cells, NALL-1 cells, ``The Journal of Immunology''
unology) J Vol. 113, 1334-134
M-7002 cells and B-7101 cells described in page 5 (1974), "Tissue Culture" Vol. 6, No. 13,
JB described on pages 527-546 (1980)
L cells, EBV-8a cells, EBV-wa cells,
EBV-HO cells, MOLT-3 cells, and other BAL
M-2 cells, CCRF-8B cells (A'rCCCCI,
120) Established lymphoblastoid 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, radiation, etc. to create culture lines. Cells that have been transformed are suitable.

In addition, the gene having the ability to produce new lymphokine II in these human-derived cells can be transferred by means of cell fusion using polyethylene glycol, Sendai virus, etc.
It can be treated with genetic recombination methods using enzymes such as NAIJ gauze, restriction enzymes (nucleases), and DNA polymerases to increase its growth rate and increase the ability to produce new lymphokine II per cell. However, the strains described herein are not limited to only Ia cells. These cells can be freely used alone or in combination of two or more types in the process up to the induced production of the new lymphokine ■, which will be described later. If necessary,
In addition, leukocytes, lymphocytes, etc. collected and prepared from humans, for example, can also be used in combination.

The warm-blooded animals used in the present invention may be those in which human-derived cells can proliferate, such as chickens, birds such as pigeons, dogs, cats, monkeys, rabbits, goats, pigs, horses, cows, Mammals such as guinea pigs, horses, horses, wildebeest rats, hamsters, regular 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 that reaction as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or still in the newborn stage. , those from childhood are preferred.

Furthermore, 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 reaction before transplantation.

If the animal used is a nude mouse or nude rat, the immune response will be weak even if the animal is fully grown.
1. Cultured human-derived cells can be transplanted without the need for these pretreatments. @, is particularly advantageous because it can multiply rapidly.

In addition, cultured human-derived cells, for example,
After transplanting the cells into mice and growing them, the cells are then transplanted into nude mice.
Furthermore, they are free to increase the amount of new lymphokines induced therefrom.

In this case, transplantation may be carried out not only between the same species and the same genus, but also between cages and 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,
A porous filtration membrane capable of blocking the passage of animal cells, such as a membrane filter with a pore diameter of 10 to 10 m.
1. Diffusion chambers of various known shapes and sizes equipped with ultrafiltration membranes or follower fibers are buried in the animal's body, for example, in the abdominal cavity, while receiving body fluids containing nutrients from the animal's body. Any of the human-derived cells cultured as described above can be grown in the chamber.

In addition, if necessary, a chamber in which the solution containing nutrients in this chamber is connected to body fluids in the animal body and perfused therein is attached to the surface of the animal body, for example, to monitor the proliferation state of human-derived cells in the chamber. It is possible to see through the window provided on the surface of the chamber, and by making it possible to remove and replace only a portion of the chamber, it is possible to proliferate the cannons for the entire lifespan without slaughtering the animal, thereby increasing the amount of cells produced per animal enclosure. can be further increased.

Methods using these diffusion chambers not only allow for easy collection of only human-derived cells since human-derived cells do not come into direct contact with animal cells, but also reduce the risk of unwanted immune reactions. There is no need for pre-treatment to inhibit the use of this method, and it has the advantage of being able to be used freely in a variety of warm-blooded animals.

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 time period for growing human-derived cells is usually 1 to 10
The goal can be achieved within a week.

It is also a finding that the number of human-derived cells obtained in this way reaches about 10 to 10 cells or more per animal.

In other words, the number of human-derived cells grown in the animal body is approximately 10 to 10 times the number of cells transplanted per animal.
The result is about 10 to 10 times, and even more than that when inoculated and propagated in an in vitro nutrient medium, which is extremely convenient for the production of new/hocaine H.

There is no limit to the method for inducing and producing new lymphocytes from the human-derived cells grown in this manner. that is,
It is also possible to cause the new lymphokine II inducer to act in the grown animal body.

For example, new lymphokine I was detected in human-derived cells grown in suspension in the ascites fluid in the peritoneal cavity, and in tumor spindles formed subcutaneously.
The new lymphokine (2) can be induced and produced by direct action of the I-inducing agent, and then the new lymphokine (2) can be purified and collected from the serum, ascites, or tumor.

Furthermore, proliferating cells derived from humans can be removed from the body of a non-human animal, and a new lymphokine (2) inducer can be applied to induce the production of new lymphokine (2) in vitro. For example, human-derived cells grown in ascites are collected, or subcutaneous tumors containing human-derived cells are excised and dispersed, and the resulting cells are placed in a nutrient medium maintained at approximately 20 to 40°C at a cell concentration. is about 105~108/rnl! The new lymphokine (2) can be induced and produced by floating the lymphokine (2) and treating it with a new lymphokine inducing agent, which can then be purified and collected.

Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells are left in the chamber or removed from the chamber and treated with a new lymphokine II inducer to induce the production of new lymphokine ■. You can also do that.

However, as mentioned above, human-derived cells obtained using warm-blooded animals other than humans may 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 (2) by applying a new lymphokine (2) inducer.

In addition, for example, after proliferating human-derived cells, the new lymphokine ■ is first induced and produced in the animal body, 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 ■ for the induction production of new lymphokine H two or more times, or embedding or connecting them in an animal body. It is also possible to further increase the amount of new lymphokine produced by pressing the animal's side on the body by changing the chamber to increase the number of cells obtained.

The new lymphokine inducer of the present invention includes viruses, nucleic acids, and
nucleotides, phytohemagglutinin, and concanavalin A, which are known as γ-interferon inducers.
, I-''-Quweed mitogen, lipopolysaccharide, endotokin, polysaccharides, bacteria, etc. are used as appropriate.Also, for sensitized cells, antigens are new/hokine inducers. be.

Furthermore, when inducing the production of new lymphokine II from human-derived cells, α-
It is also possible to increase the production amount of the new lymphokine (2) by using an interferon inducer and a γ-interferon inducer together.

Furthermore, it was found that not only the new lymphokine (■) was produced by these induced productions, but also human interferon, which is highly species-specific, was also produced at the same time.

This makes it possible to simultaneously produce two or more valuable human physiologically active substances12, and also to make advanced use of human-derived cells possible, from the point of view of supplying new lymphokines■ and human interferon in large quantities at low cost. This is extremely convenient.

The new lymphokine ■ induced and produced in this way can be easily purified and separated and collected by performing known sheath type separation methods, such as salting out, dialysis, filtration, centrifugation, concentration, and freeze-drying. I can do it. 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, it is possible to collect new lymphokines of the highest purity. is also possible.

In addition, a warm-blooded animal other than humans is immunized with the new lymphokine (■) obtained in this way as an antigen, antibody-producing cells are collected from the animal, and these cells are fused with myeloma cells. A fused cell having the ability to produce an anti-lymphokine antibody is selected from the fused cells, the selected cell is grown, and the produced monoclonal antibody is reacted with, for example, bromcyane-activated sepharose using an immobilized monoclonal antibody obtained. It can also be advantageously used to purify and collect highly pure new lymphokine (2) in high yield.

The new lymphokine ■ purified and manufactured in this way is
The physical and chemical properties are ■Molecular weight 20, OOO±2,000 ■Isoelectric point pI = 6.2±0.3 ■Mobility Disc -)'AGE, R, f = 0.29±0.0
2 ■ Ultraviolet gland absorption spectrum Maximum absorption around 280 nm ■ Soluble in solvent Soluble in water, physiological saline or phosphate buffer Ethyl ether, ethyl acetate Still slightly soluble or insoluble in chloroform ■ Color reaction Lowry method Or shows a positive protein reaction by the microburet method and a positive barrier reaction by the phenol-sulfuric acid method or the anthrone-sulfuric acid method.It shows cytotoxic activity against L929 cells, and has cytostatic activity and interferon activity against KB cells. Active stability in a water bath solution Stable up to (a) ℃ by holding at pH 7.2 for 16 hours; pH 4.0 to 11.0 by holding at 4℃ for 16 hours. Stable in the O range■ It was found to be stable for more than one month when stored frozen at -10°C.

It has also been found that the new lymphokine (2) has the ability to damage and kill not only mouse L929 cells but also many human tumor cells, but does not substantially damage normal human cells.

Therefore, the new lymphokine II can be advantageously used as a preventive or therapeutic agent for new lymphokine II-sensitive diseases, such as various malignant tumors, in the form of a composition containing it.

In particular, the present invention provides enhanced anti-tumor proboscis against chemotherapeutic agents containing the new lymphokine ① as an active ingredient, reducing the dose of chemotherapeutic agents by approximately 1/2 to 1/10.
This not only suppresses the occurrence of side effects but also broadens the antitumor spectrum, making it possible to treat drug-resistant tumors.

The chemotherapeutic agents used in the present invention include, for example, melphala/, tucrophosphamide, ifosfamide, estramustine sodium phosphate, busulfan, improsulfan tosylate, N-methyl-3,3'-dimesyloxy Alkylating agents such as dipropyramine, biphenyl-4,4'-thisul7onate, carbocone, thiotepa, carmustine, nimustine hydrochloride, streptozocin, dacarbazine, bipopromane, methotrexate, fluorouracil, tegafur, carmofur, cytarabine, ancitabine hydrochloride, enocitapine , mercaptopurine, antimetabolites such as thioinosine, Dokinrubicin/, daunorubicin, aclarubicin, pleomycin, hefuromycin, mitomycin C1 actinomycin D1
Antitumor antibiotics such as actinomycin C1 chromomain A3, mithramycin, neocarzinostatin,
There are plant alkaloids such as vincristine sulfate, vinblastine sulfate, vindesine sulfate, and podophyllotoxin, and one or more of these may be used as appropriate. Furthermore, in the case of prostate cancer, breast cancer, etc., antitumor hormones such as gredonizolo/, methyltestosterone, and conjugated estrogen may be appropriately used in combination, if necessary.

The method of combining these chemotherapeutic agents and the new lymphokine (2) does not need to be limited to specific conditions and can be selected as appropriate.

The activity of the new lymphokine ■ is influenced by KB cells as target cells,
It was measured using L929 cells.

That is, when using KB fine foam, "Cancer Chemotherapy Lipo-7"
rapyReports) Parts
3 J Volume 3, No. 2, September 197
2, the growth inhibitory activity of K B m cells was measured, and when L929 cells were used, Evic (E
, Pick), [Tsumoa Necrosis Factor
In Lymphokines (Tumor Necrosis)
Factor in Lymphokines)
J Vol. 2, pp. 245-249, Academic Press, x
(Academic)'ress) (1981) in the presence of actinomycin D.
The cytotoxic activity exerted on 29 cells was measured.

In this specification, unless otherwise specified, an activity measurement method using L929 cells was adopted.

The activity of interferon, which is highly species-specific to humans, is described in "Protein Nucleic Acid Enzyme" Vol. K, No. 6, pp. 616-643 (1).
It was measured by the known plaque half-life method using human amnion-derived FL cells as reported in 1997).

Red blood cell C suspected collection is
alk), [The Journal of Immunology (
The Journal of Immunology
) J Vol. 49, p. 87 (1944).

Next, we will explain the new lymphokine ■ through experiments.

Experiment A-1 Preparation of Partially Purified New Lymphokine ■ Neonatal hamsters were injected with antiserum prepared from rabbits by a known method to weaken the immune response of the hamsters, and then HALL-1, J cells were injected subcutaneously into the hamsters. 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 cultured in serum-supplemented PMI 1640 medium (pH 7,2).
) and suspended in the same medium to a concentration of approximately 2 x 10 /mA'. For this cell suspension, about 4
Add Sendai virus with 00 hemagglutination titer and heat at 37°C.
The cells were kept for 24 hours to induce the production of new lymphokine ■.

This was centrifuged at about 4°C and about i, ooo g to remove the precipitate, and the resulting supernatant was centrifuged at about i, ooo g.
The filtrate obtained by dialysis with physiological saline containing MIJ phosphate buffer and microfiltration was applied to an antibody column on which anti-interferon antibodies were immobilized, and the non-adsorbed fraction was collected. Further, the active fraction was collected by chromatography, concentrated, and lyophilized to obtain a powder containing the new lymphokine II activity.

The specific activity of this powder was approximately 106 units/m9 protein. Furthermore, the yield of the new lymphokine ■ was approximately 20 million units per hamster.

Experiment A-2 Preparation of Anti-French Lymphokine ■Antibodies The new lymphokine ■ obtained by the method of Experiment A-1 was dissolved in physiological saline to a protein concentration of approximately 0.05 w/v%, and Freund's complete Mix equal amounts of adjuvant emulsion and inject 0.2rnl of this mixture subcutaneously into mice.
A day later, the mice were immunized again with the same injection. Spleen cells and mouse myeloma cells P3-X63-Ag are obtained by inducing and producing anti-lymphokine antibodies in cells that have the antibody-producing ability, removing the spleen from this mouse, and dispersing it into small pieces.
8 “Flow Laboratories”
tories) into a 50 W/V% polyethylene glycol-1000 solution (pH 7.2, temperature 37°C) prepared with serum-free Eagle's minimal basic medium.
After maintaining the suspension for 5 minutes at a concentration of 10 cells/me, the cells were diluted 1-fold with the above-mentioned basic medium, and then the cellulose cells were diluted with the above-mentioned basic medium, and then the cells were incubated with Somatic Cell Genetox, as described by Davison et al.
ics) J Vol. 2, pp. 175-176 (1976) to collect fused cells that can proliferate in a hypoxanthine-aminopterin-thymidine culture solution,
From these fused cells, fused cells capable of producing anti-lymphokine (III) antibodies were selected. Approximately 10 of the obtained fused cells were transplanted into the peritoneal cavity of each mouse and raised for two weeks, after which the mice were sacrificed and body fluids such as ascites and blood were collected and centrifuged.
This supernatant was salted out with ammonium sulfate to collect a precipitated fraction with a saturation level of 30-50%, which was then dialyzed, and the new lymphokine ■ obtained by the method of Experiment A-1 was added to this solution with bromcyane-activated sepharose. Affinity chromatography was performed using the immobilized new lymphokine II gel obtained by reaction at room temperature to obtain anti-refractory lymphokine ■ antibody fraction, which was dialyzed, concentrated, and freeze-dried to obtain new lymphokine H monoclonal antibody powder. was collected.

Quartz crystals showed 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 A partially purified product of new lymphokine ■ prepared by the method of Experiment A-1 was prepared using the method of Experiment A-2. Perform affinity chromatography using a gel immobilized with and collect the active fraction of the new lymphokine ■.
Dialyzed, concentrated and lyophilized.

This product is a highly purified new lymphokine (■), and its specific activity was approximately 109 units/■ protein.

The physical and chemical properties of this product were investigated.

■Molecular weight K Weber & M Osbor 7 (K,
Weber and M, '0sborn), [Journal Off Biological Chemistry (Jour
nalof Biological Chemistry
y) It was investigated by 5DS-polyacrylamide gel electrophoresis according to the description in J Vol. 244, p. 4406 (19f39). That is, in the presence of 0.1% SDS, about 10μ2 of the sample was loaded onto a 10% acrylamide 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.

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

■ Electrical Mobility B.J. Davis, [Annu@Is of the New York Academy of Sciences]
York Academy of 5 Sciences
) J Vol. 121, p. 404 (1964), apply approximately 10μ of the sample to a 7.5% acrylamide gel column.
? After loading and electrophoresis at pH 8.3 and 3 mA per column for 2 hours, it was extracted and the electrical mobility was determined from the activity measurement, and it was found to be fO129±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 using 2
It showed maximum absorption around 8 Onm. (2) Solubility in solvents Soluble in water, physiological saline or phosphate buffers Slightly soluble to insoluble in ethyl ether, ethyl acetate or chloroform.

■ A positive protein reaction was shown by the color reaction Lory method or the microbead method, and a positive reaction was shown by the phenol-sulfuric acid method and the anthrone sulfuric acid method.

■Effect: It exhibited cytotoxic activity against L929 cells. Substantially no cytostatic activity or interferon activity against KB cells was shown.

■Activity stability in aqueous solution ■) Thermal stability A sample of approximately 1 x 10 units/rnl was prepared at pi-1 at each temperature.
After holding at 7.2 for 30 minutes, the remaining activity was measured and found to be stable up to 60°C.

2) Add 0.1 ml of a sample with a pH stability of approximately 1 x 10 units/ml to each pH buffer (pH 2-7-myclevein buffer fi, (Mc
llvainebuf fer), pH 7-8-...
phosphate buffer
, pH 8-11 ・Glycine-NaOH buffer) 1
This 0.1
ml with pH 7, 2,0,05M phosphate buffer
As a result of measuring the remaining activity after adjusting the pH to 4,
It was stable in the range of 0 to 11.0.

3) Stability against DISPASE: Approximately 1 x 10 units/ml of sample was injected with 1 dispase (protease derived from Bacillus bacterium, produced by Joint Alcohol Co., Ltd.).
00 units/me, pH 7.2, temperature 3.
The reaction was carried out at 7° C. for 0 to 2 hours, sampled economically, and the reaction was stopped by adding bovine serum albumin to lw/v%. As a result of measuring the residual activity of new lymphokine (■) in this solution, it was found that new lymphokine (2) was unstable due to disbase treatment and the activity of new lymphokine (2) 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■ is a previously known lymphotoxin, Tsumoa necrosis factor (T
It has clearly different physicochemical properties from lymphokines such as NF) and interferon.

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

10 cells of each type of human-derived cells were placed in a known nutrient medium 1rIL1 supplemented with fetal bovine serum, and after culturing for 1 day, the new lymphokine ■ prepared by the method of Experiment A-1 or Experiment A-3 was added. 0.1 ml of physiological saline containing 50 units or 500 units was added and cultured at 37°C for 2 days.

After culturing, “Applied Microbiology (Ap
pl ied Microbiology ) J No. n
vol., No. 4, pp. 671-677 (1971), live cells were stained with the stain Neutral Red, the stain was then eluted with andethanol, and the eluate The amount of living cells was measured from the absorbance at 540 nm.

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

Cell proliferation inhibition rate % was calculated from the following formula.

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

As is clear from the results shown in Table 1, the new lymphokine ■ had almost no effect on normal cells, and was found to significantly suppress the proliferation of various malignant tumor cells. However, it has been found that the effect can be obtained 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. , 100 or 1,000 single-position □Q were intravenously injected once daily for 15 days. Thereafter, mice were sacrificed and tumor weights were measured.

The results are shown in Table 2.

Rice The risk rate is 5% or less, and there is a 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 nude mouse derived from BALB/C, and from the time when the tumor volume reached approximately 200 mJ, the method of Experiment A-1 or Experiment A-3 was carried out. The obtained new lymphokine (■) dissolved in physiological saline was intravenously injected once daily at a dose of 9 to 100 units/1,000 units. Thereafter, the nude mice were sacrificed and the weight of the tumor was measured.

The results are shown in Table 3.

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

Experiment B-4 We conducted an acute toxicity test of the new lymphokine ■ obtained by the method of Experiment A-3 using postnatally stressed mice, and found that the toxicity of the new lymphokine ■ was extremely low when injected intraperitoneally. LD5o was found to be greater than 10 units.

As is clear from the above experiments, the new lymphokine (■) of the present invention is effective in suppressing the growth of malignant tumors not only in vitro but also in vivo, and is stable considering its effective dose. is extremely high.

The daily dose for adults of the new lymphokine ■ 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 units,
000 units, 10 to 50,000,000 units for transdermal or transmucosal applications such as ointments or suppositories, and 50 to ioo, ooo, ooo for systemic injections such as intravenous and intramuscular injections.
unit, 500 to 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. Considering the toxicity, effective amount and safety of the new lymphokine H, it is desirable that the drug used contains 5 or more units of the new lymphokine (I) per pharmaceutical preparation.

The form of the new lymphokine II-sensitive disease preventive or therapeutic agent containing the new lymphokine (2) can be freely selected depending on the purpose.

Orally administered preparations include enteric-coated preparations such as capsules, tablets, and powders; rectal suppositories are used as intrarectal torsion preparations; and injection preparations include, for example, freeze-dried preparations that are dissolved in distilled water for injection. It can also be used as an injection, other nasal or eye drops, and an ointment.

In addition, when treating malignant tumors using new lymphokine I, 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 returning it to the patient's body, this malignant tumor can be treated more effectively.

In addition, new lymphokines along with other lymphokines such as interferon, TNF, lymphotoxin, TCGF, alkylating agents, antimetabolites, anti-tumor antibiotics, chemotherapeutic agents such as plant associate alkaloids, etc. It can also be advantageously used in combination with other drugs to further enhance the antitumor effect.

In particular, a new lymphokine ■ was found to enhance the antitumor effects of chemotherapeutic agents.

Next, we will explain the antitumor effect-enhancing effect of the new lymphokine (■) through experiments.

Experiment C-1 Effect of new lymphokine (■) on enhancing the antitumor effect of various chemotherapeutic agents Using malignant tumor cells, the effect of new lymphokine (2) on enhancing the antitumor effect of various chemotherapeutic agents was investigated.

Ten human-derived malignant tumor cells were added to 1 ml of a known nutrient medium supplemented with fetal bovine serum, and cultured for one day.
To this was added 0.1 ml of physiological saline containing 100 units of Lymphokine H prepared and trapped by the method of Experiment A-'3 and/or a chemotherapeutic agent, and cultured at 37°C for 2 days.

For control experiments, a new lymphokine ■ and saline containing no chemotherapeutic agent were used.

After completion of the culture, the amount of viable cells was measured according to the method of Mitsu@B-1, and the cell growth inhibition rate (%) was determined.

In addition, the concentration of chemotherapeutic agent is culture e, salt per ml! Nimustine (ACNU) 1.0X10 L Fluorouracil (5-FU) 1.5X10 g, Doxorubicin (ADM) 1.0X10 g, Mitomycin C (M
MC) 2.5xlOg and sulfuric acid pink') Sf:y
(VCR) 1.5×10 g.

The results are shown in Table 4.

As is clear from the results in Table 4, the new lymphokine ■ significantly enhances the antitumor effects of various chemotherapeutic agents.

Experiment C-2 A piece of human breast cancer tissue was subcutaneously transplanted into the back of a BALB/C-derived nude mouse, and from the time when the tumor volume reached approximately 200 cm, new cancer tissues obtained by the method of Experiment A-3 were obtained. U
And/or mitomine 7C dissolved in physiological saline was intravenously injected once daily during the loading period.

Thereafter, the nude mice were sacrificed and the weight of one tumor was measured.

Note that, in the control experiment, a physiological saline solution containing no new lymphokine (■) and a chemotherapeutic agent was used.

The results are shown in Table 5.

Rice The risk rate is 5% or less, and there is a statistically significant difference compared to the control value.

As is clear from the results in Table 5, the new lymphokine ■ significantly enhances the antitumor effect of chemotherapeutic agents against malignant tumors even in in vivo tests.

From the results of Experiment C described above, even when using low-concentration chemotherapeutic agents with insufficient anti-tumor effects, high anti-tumor effects can be achieved by using the new lymphokine of the present invention in combination. It has been found.

By using New/Hocaine■ as an antitumor effect enhancer of chemotherapy agents, the concentration of chemotherapy agents used can be significantly lowered, not only suppressing the occurrence of side effects, but also significantly expanding the antitumor spectrum. be able to.

The antitumor effect enhancer containing the new lymphokine ① of the present invention exhibits an antitumor effect when used together with a chemotherapeutic agent.

The antitumor effect enhancer containing the new lymphokine ■ and the chemotherapeutic agent may be mixed in advance and administered simultaneously by the same route, or one of them may be administered first, followed by the other. A method of administering the same route or another route can be selected as appropriate.

Hereinafter, Example A will describe a production example of the new lymphokine (2) of the present invention, and Example B will describe a production example of an antitumor effect enhancer containing the new lymphokine (2) of the present invention.

Example A-1 Human-derived lymphoblastoid BALL-1 cells were cultured in Eagle's minimal basal medium (pH 7) supplemented with fetal bovine serum.
, 4), 37. In vitro (1 nv
The cells were cultured in suspension on itro). The obtained mud cells were washed with serum-free Eagle's minimal basal medium (pH 7.4),
It was suspended in the same medium at a concentration of about 1X10/ml. Approximately 1.0% of Sendai virus per rnl was added to this suspension. OOO
Red blood cell agglutination was added and kept at 31°C for 1 day to induce the production of new lymphokine (■). This was centrifuged at 4°C and approximately 1,000 g, and the resulting supernatant was diluted to pH 7,2,0,01M.
! The filtrate obtained by dialysis with physiological saline containing 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 used in the experiment. It was purified by affinity chromatography using a monoclonal antibody gel column in the manner described in A-3, and then concentrated to obtain a concentrated solution of the new lymphokine (1) having a specific activity of approximately 109 units/m9 protein.

The activity yield was approximately 1.5 million units per 1 suspension during induction production.

Example A-2 Neonatal hamsters were injected with an antiserum prepared from rabbits using a known method to weaken the gnome star's immune response, and then human-derived lymphoblastoid cells were subcutaneously cultured. Cells BALL-1 cells were transplanted, and then 3
They were kept for a week. A single aneurysm weighing approximately 15 g that had occurred under the skin was removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were cultured in serum-free R1'MI 1640 medium (pH 7,
2) and suspended in the same medium at approximately 5×10 6 /me. Approximately 1.0 ml of Sendai virus was added to this suspension.
00 hemagglutination titer and E. coli-derived endotoxin at a concentration of approximately 10 μm/nl were added and kept at 37° C. for 1 day to induce the production of new lymphokine (2). This is about 4
Centrifuge at approximately 1,000 g at °C to remove the precipitate.
The obtained supernatant was dialyzed for 21 hours with physiological saline containing a pH 7, 2, 0, 0,01M 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 new lymphokine H having a specific activity of approximately 109 units/m9 protein.

The activity yield was about 32 million units.

Example A-3 Adult nude mice were intraperitoneally transplanted with cultured human-derived lymphoblastoid cells, TALL-1 cells, and then raised in a conventional manner for 5 weeks. Into this abdominal cavity, about 3,000
Two-casosul disease virus with a hemagglutination titer of 0 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 new lymphokine (2).

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 function of the mouse, human-derived lymphoblastoid cells Mono were cultured subcutaneously in the mouse.
-1 cells were transplanted and then reared for 3 weeks in the usual manner. After removing a subcutaneous tumor weighing approximately 10 g, the cells were dispersed in the same manner as in Example A-2. After suspending the cells in the same manner as in Example A-2, approximately 500 hemagglutination titer per me of Sendai virus and 0.8 μ2 per ml of concanavalin A were added to this suspension, and the mixture was kept at 37°C for 1 day. The new lymphokine ■ 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 the new lymphokine (■).

The activity yield is 1 mouse, 62. ooo million units Example A-5 Human-derived lymphoblastoid Namalva cells cultured in the same manner as in Example A-2 were transplanted into newborn hamsters, and then cultured in a normal manner for 4 hours. They were kept for a week. A subcutaneous tumor weighing approximately 20 g was loosened in the same manner as in Example A-2 to obtain a cell suspension of approximately 3×10/m/m. Sendai virus was added to this suspension at a hemagglutination value of approximately 1,000 per rrLt, and the suspension was kept at 20°C for 2 days to induce the production of new lymphokine (2).Then, it was purified and concentrated in the same manner as in Example A-1 to produce new lymphokine (2). A concentrated solution was obtained.

The activity yield was approximately 22 million units per hamster.

Example A-6 Cultured human-derived lymphoblastoid cells N were placed in a 10 ml plastic cylindrical diffusion chamber equipped 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 the human-derived cells thus obtained was approximately 5XlO/mA', which was found to be approximately 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 this suspension was added with rt.
Niecastle disease virus with a hemagglutination titer of about 500 tl was added after inactivating most of it with ultraviolet rays, and phytohemagglutinin was added with a hemagglutination titer of about 500.
The new lymphokine ■ was induced to be produced for several days. Thereafter, it was purified in the same manner as in Example A-2, concentrated and dried to obtain a powder of new lymphokine (2).

The activity yield was about 8 million units for 81 rough animals.Example A-7 Fertilized chicken eggs K kept at 37°C for 5 days, human-derived cell line CCRF-CE, and M cells were transplanted. After that, 37
It was kept at ℃ for 1 week. After the eggs were broken, proliferating cells were collected. The cells were grown at 5×10/m in the same manner as in Example A-1.
It was suspended in A'. Sendai virus with a hemagglutination titer of about 500 per M was added to this suspension and kept at 37°C for 1 day to induce the production of new lymphokine ■, and then Example A-
It was purified in the same manner as in 2 and concentrated and dried to obtain a powder of new lymphokine (2).

The activity yield was approximately 700,000 units per 10 fertilized eggs.
00,000 units dissolved in 200 ml of saline,
Filter aseptically using a membrane filter.

The saline solution is filled into sterilized glass containers in an amount of 2 Inl and freeze-dried, and the containers are sealed tightly to obtain a freeze-dried powder preparation.

This product is suitable for the treatment of breast cancer, lung cancer, liver cancer, leukemia, etc. as an antitumor effect enhancer of chemotherapeutic agents such as melphalan, mentrexate, and doxorubicin.

Example B-2 Injection In the production of the injection solution of Example B-1, 300,000,000 units of α-interferon derived from lymphoblastoid cells were added to 200 ml of physiological saline together with 500,000 units of new lymphokine n. Example B-1 except that it was dissolved in
A lyophilized powder formulation was obtained in the same manner as above.

This product is used as an antitumor effect enhancer of chemotherapeutic agents such as tegafur, mitomycin C1 vincristine sulfate, etc., and is used as the injection solution of Example B-1 for various malignant salivary tumors such as breast cancer, lung cancer, liver cancer, gastric cancer, and leukemia. It exhibits a remarkable therapeutic effect compared to other drugs.

Example B-3 Ointment The new lymphokine ■ prepared by the method of Example A-'3 was added to a small amount of liquid paraffin according to a conventional method, and then vaseline was added to make an ointment with a concentration of 20,000 units/9.

This product is suitable for the treatment of skin cancer, breast cancer, lymphoma, etc. as an antitumor effect enhancer of chemotherapeutic agents such as cyclophosphamide, fluorouracil, and vinblastine sulfate.

Example B-4 points Eye preparation Distilled water sooml β-phenylethyl alcohol 5m
E and the new lymphokine (2) prepared by the method of Example A-4 were added with common salt to make it isotonic to 20,000,000 units, and then diluted with distilled water to 1,000 units per eye drop.

Quartz is suitable as an antitumor effect enhancer for chemotherapeutic agents such as cytarabine and vinblastine sulfate, and for the treatment of retinoblastoma and the like.

Example H-5 Enteric tablets When the new lymphokine ■ prepared by the method of Example A-7 is tableted using a mixture of starch and maltose according to a conventional method, one tablet (100 tablets) of the new lymphokine ■ is prepared using a mixture of starch and maltose. ) hit 2
00,000 units to produce tablets, which were then coated with methylcellulose phthanate to form enteric-coated tablets.

As an antitumor effect enhancer of chemotherapeutic agents such as crystal ha, doxorubicin, fluorouracil, and mitomycin C,
Suitable for treatment of colorectal cancer, colon cancer, liver cancer, etc.

Claims (4)

[Claims] (1) Physical and chemical 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] An antitumor effect enhancer containing a new lymphokine II that is stable for one month or more when stored frozen at -10°C. (2) New lymphokine II is produced from human-derived cells that have the ability to produce new lymphokine II, transplanted into the body of a warm-blooded animal other than humans, or cells that are grown while being supplied with body fluids from that warm-blooded animal. The antitumor effect enhancer according to claim (1), which is obtained by purifying and collecting the antitumor effect enhancer. (3) The antitumor effect enhancer according to claim (1) or (2), characterized in that the antitumor effect enhancer contains another lymphokine together with the new lymphokine II. (4) The antitumor effect enhancer according to claim (1), (2), or (3), wherein the other lymphokine is interferon.